Fractionation of oil-bearing microbial biomass

ABSTRACT

The invention generally relates to the production of hydrocarbon compositions, such as a lipid, in microorganisms. In particular, the invention provides methods for extracting, recovering, isolating and obtaining a lipid from a microorganism and compositions comprising the lipid. The invention also discloses methods for producing hydrocarbon compositions for use as biodiesel, renewable diesel, jet fuel, and other materials.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Application No. 61/181,252, filed May 26, 2009, which isincorporated herein by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing, appended hereto as pages1-45.

FIELD OF THE INVENTION

The invention generally relates to the production of oil compositions,such as a lipid, in microorganisms. In particular, the inventionprovides methods for extracting, recovering, isolating and obtaining alipid from a microorganism and compositions comprising the lipid. Theinvention also discloses methods for producing hydrocarbon or lipidcompositions for production of biodiesel, renewable diesel, jet fuel,and lipid surfactants having various carbon chain lengths, including C8,C10, C12 and C14.

BACKGROUND OF THE INVENTION

Fossil fuel is a general term for buried combustible geologic depositsof organic materials, formed from decayed plants and animals that havebeen converted to crude oil, coal, natural gas, or heavy oils byexposure to heat and pressure in the earth's crust over hundreds ofmillions of years.

Fossil fuels are a finite, non-renewable resource. With globalmodernization in the 20th and 21st centuries, the thirst for energy fromfossil fuels, especially gasoline derived from oil, is one of the causesof major regional and global conflicts. Increased demand for energy bythe global economy has also placed increasing pressure on the cost ofhydrocarbons. Aside from energy, many industries, including plastics andchemical manufacturers, rely heavily on the availability of hydrocarbonsas a feedstock for their manufacturing processes. Alternatives tocurrent sources of supply could help mitigate the upward pressure onthese raw material costs.

Lipids for use in biofuels can be produced in microorganisms, such asalgae, fungi, and bacteria. Typically, manufacturing a lipid in amicroorganism involves growing microorganisms, such as algae, fungi, orbacteria, which are capable of producing a desired lipid in a fermentoror bioreactor, isolating the microbial biomass, drying it, andextracting the intracellular lipids.

There is a need for a process for extracting lipids from microorganismwhich solves the above problems of low efficiency and high cost of lipidextraction from microorganism. The present invention provides a solutionto these prior art problems.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a method ofextracting a lipid from a microorganism. In one embodiment, the methodcomprises lysing a cultured microorganism that has not been subjected toa drying step between culturing and lysing, and which contains a lipid,to produce a lysate, treating the lysate with an organic solvent for aperiod of time sufficient to allow the lipid from the microorganism tobecome solubilized in the organic solvent, and separating the lysateinto layers comprising a lipid:organic solvent layer and an aqueouslayer, whereby the lipid is extracted from the microorganism.

In another aspect, the present invention is directed to a compositioncomprising a lipid isolated from a microorganism, and an oil obtainedfrom a source other than the microorganism. In some cases, the ratio ofthe lipid to the oil is between 1 and 100. In other cases, the ratio ofthe lipid to the oil is between 1 and 10. In some embodiments, the lipidcomprises at least 50% of a C18:1 lipid, or at least 10% of a C12 andC14 lipid combined.

In another aspect, the present invention is directed to a method ofproducing biodiesel. In one embodiment, the method comprises lysing alipid-containing microorganism to produce a lysate, treating the lysatewith an organic solvent for a period of time sufficient to allow thelipid from the microorganism to become solubilized in the organicsolvent, separating the lysate into a lipid:organic solvent compositionand an aqueous composition and, optionally, an emulsified compositionand/or cell pellet composition, removing the lipid:organic solventcomposition from the aqueous composition, emulsion composition, or cellpellet composition, and transesterifying the lipid:organic solventcomposition to produce biodiesel. In some cases, the biodiesel meets orexceeds the ASTM D6751 biodiesel standard and/or the EN 14214 biodieselstandard. In one embodiment, the microorganism is not subjected to adrying step.

In some embodiments of the biodiesel production method at least 20% w/vof total lipid from the microorganism is C18:1. In one embodiment, thelayers further comprise a lipid:aqueous emulsion layer and/or a cellpellet. Transesterification of the lipid:organic solvent can beperformed without substantially separating the lipid from the organicsolvent.

In still another aspect, the present invention is directed to a methodof producing renewable diesel. In one embodiment, the method compriseslysing a lipid-containing microorganism to produce a lysate, treatingthe lysate with an organic solvent for a period of time sufficient toallow the lipid from the microorganism to become solubilized in theorganic solvent, separating the lysate into a lipid:organic solventcomposition and an aqueous composition and, optionally, an emulsifiedcomposition and/or cell pellet composition, removing the lipid:organicsolvent composition from the aqueous composition, emulsion composition,or cell pellet composition, and treating the lipid:organic solventcomposition to produce a straight chain alkane renewable diesel product.In some cases, the renewable diesel meets or exceeds the ASTM D 975standard.

In various embodiments of the renewable diesel production method, themicroorganism has not been subjected to a drying step. In some cases,treating the lipid:organic solvent composition is performed withoutsubstantially separating the lipid from the organic solvent. In someembodiments, treating the lipid:organic solvent composition compriseshydrotreating, hydroprocessing, or indirect liquefaction.

In another aspect, the present invention is directed to a method ofproducing a jet fuel. In one embodiment, the method comprises lysing alipid-containing microorganism to produce a lysate, treating the lysatewith an organic solvent for a period of time sufficient to allow thelipid from the microorganism to become solubilized in the organicsolvent, separating the lysate into a lipid:organic solvent compositionand an aqueous composition and, optionally, an emulsified composition orcell pellet composition, removing the lipid:organic solvent compositionfrom the aqueous composition, emulsion composition, or cell pelletcomposition, treating the lipid:organic solvent composition to produce astraight chain alkane, and cracking the straight chain alkane to producethe jet fuel product.

In various embodiments of the jet fuel production method, themicroorganism has not been subjected to a drying step. In some cases,treating the lipid:organic solvent composition is performed withoutsubstantially separating the lipid from the organic solvent. In someembodiments, treating the lipid:organic solvent composition is performedby flowing the lipid:organic solvent composition to a fluid catalyticcracking zone, and can further comprise contacting the lipid:organicsolvent composition with a catalyst at cracking conditions. In oneembodiment, treating the lipid:organic solvent composition is performedby hydrodeoxygenating the lipid:organic solvent composition. In somecases, the method further comprises subjecting the hydrodeoxygenatedlipid:organic solvent composition to isomerization.

In another aspect, the present invention is directed to a method ofextracting lipid from a microorganism by contacting a microorganismcontaining a lipid with an acid to produce a lysate, separating thelysate into layers comprising an aqueous layer and a lipid:aqueousemulsion layer, and extracting lipid from the emulsion layer.

In various embodiments of the method of extracting lipid from amicroorganism, the microorgansism is contacted with the acid to producean acid concentration of 5-200 mN, and contacting the microorganism canbe performed above 25° C. In various embodiments, an organic solvent isadded to the microorganism or lysate before, simultaneously with, orafter contacting the microorganism with the acid. In some cases, themicroorganism is contacted with an acid at a pH of no more than 4, at apH of no more than 3, or at a pH of no more than 2. In some embodiments,in addition to contacting the microorganism with the acid, one or moreadditional methods of lysing the microorganism is also utilized. In somecases, contacting the microorganism with the acid is performed at atemperature of 50-160° C. In other cases, the step of contacting themicroorganism with the acid is performed at a temperature of 20-65° C.In one embodiment, the lipid is extracted from the emulsion bycontacting the emulsion with an organic solvent, whereby the lipidpartitions from the emulsion into the organic solvent. In some cases,separation of the lysate is performed by cooling the emulsion below 25°C., below 10° C., or to a temperature at or below 0° C., whereby a lipidlayer separates from the emulsion layer. In one embodiment, the methodfurther comprises centrifuging the emulsion after the cooling toseparate the lipid layer. In some embodiments, the method furthercomprises separating the emulsion from the aqueous layer beforeseparating the lipid from the emulsion. In one embodiment, the lipid isextracted from the lipid:aqueous emulsion without use of an organicsolvent.

In another aspect, the present invention is directed to a method ofextracting lipid from a microorganism by lysing a microorganismcontaining a lipid to produce a lysate comprising a lipid:aqueousemulsion, and cooling the emulsion below 25° C. to separate the lipidfrom the emulsion.

In another aspect, the present invention is directed to a method ofextracting lipid from microbial biomass generated by culturing amicroorganism that produces a lipid. Extraction comprises lysing themicroorganisms in the biomass to produce a lysate comprising alipid:aqueous emulsion. In some cases, separation of a lipid layer fromthe emulsion comprises destabilizing the emulsion with the addition of asurfactant to the emulsion. In some cases, the lysate can be treatedwith an organic solvent for a period of time sufficient to allow thelipid from the microorganism to become solubilized in the organicsolvent, and the lysate can be separated into two or more layers,including a lipid:organic solvent layer and at least one aqueous layer.In some cases, the method can further include removing the lipid:organicsolvent composition from the other layer(s). Optionally, removing thelipid:organic solvent composition from the other layer(s) can beperformed without substantially separating the lipid from the organicsolvent. In some cases, the method can further include transesterifyingthe lipid:organic solvent composition to produce a fatty acid alkylester, or treating the lipid:organic solvent composition to produce astraight chain alkane. In the latter case, the method can furtherinclude cracking the straight chain alkane.

In another aspect, the present invention is directed to a composition offatty acid esters comprising esters derived from a microorganism, andesters derived from a nonmicrobial oil. In some embodiments, no morethan 20%, no more than 10%, or no more than 6%, of the esters in thecomposition are derived from a nonmicrobial oil. In some cases, theesters are selected from the group consisting of methyl, ethyl and alkylesters. In some embodiments, the cultured microorganism has not beenseparated from liquid medium used to culture the microorganism, and/oris present at a ratio of less than 1:1 v/v of cultured microorganism toextracellular liquid media.

In various embodiments of the method of extracting lipid from amicroorganism, the emulsion is cooled below 10° C., or to a temperatureat or below 0° C. In one embodiment, the method further comprisescentrifuging the lysate to produce layers comprising the emulsion and anaqueous layer. In some cases, the method further comprises separatingthe emulsion from the aqueous layer. In one embodiment, the lipid isextracted from the lipid:aqueous emulsion without use of an organicsolvent. In some cases, the method further comprises treating the lysatewith an organic solvent for a period of time sufficient to allow thelipid from the microorganism to become solubilized in the organicsolvent.

Microorganisms useful in accordance with the present invention can beselected from the group of microorganisms consisting of a bacterium, acyanobacterium, a eukaryotic microalgae, an oleaginous yeast, and afungus. In some cases, the microorganisms can be selected from Tables 1,2 or 3. Such microorganisms include microorganisms of the genusChlorella. In one embodiment, the microorganism is Chlorellaprotothecoides. In some cases, the microorganisms of the presentinvention produce a lipid that comprises at least 10% w/v of totalcellular lipid as C18 triacylglycerols, or at least 10% w/v of totalcellular lipid as C16 triacylglycerols. In some cases, themicroorganisms produce a lipid that comprises at least 10% w/v of totalcellular lipid as C14 triacylglycerols, at least 10% w/v of totalcellular lipid as C12 triacylglycerols, or at least 10% w/v of totalcellular lipid as C10 triacylglycerols. In some cases, themicroorganisms of the present invention have at least 85% 23S rRNAgenomic sequence identity to one or more sequences selected from thegroup consisting of SEQ ID NOs:7-33.

In some cases, the microorganism is of the genus Prototheca. In oneembodiment, the microorganism is Prototheca moriformis. In some cases,the microorganisms of the present invention have at least 75% sequenceidentity to one or more sequences selected from the group consisting ofSEQ ID NOs: 33, 34, 35, 16, 36, 17, 37, 38, 39, and 32.

In various embodiments in accordance with the present invention, lysingthe cultured microorganisms is performed at an acidic pH, at a pH of nomore than 5, at a pH of no more than 4, at a pH of no more than 3, or ata pH of no more than 2. In some embodiments, lysing the culturedmicroorganisms is performed at a pH of at least 9. In some cases, lysingthe cultured microorganism comprises one or more methods of lysingselected from the list consisting of heating, sonication, mechanicallysis, osmotic shock, pressure oscillation, expression of an autolysisgene, exposure to pH above 8, exposure to pH below 6, and digestion withan enzyme. In some cases, lysing the microorganism comprises acidiclysis and heating. In one embodiment, the microorganism is lysed bydigestion with a polysaccharide-degrading enzyme, which can be apolysaccharide-degrading enzyme from Chlorella or a Clorella virus. Insome cases, the microorganism is lysed by digestion with a protease. Inother cases, the microorganism is digested with a combination of atleast one protease and at least one polysaccharide-degrading enzyme. Insome cases, the protease is alcalase and/or the polysaccharide-degradingenzyme is mannaway. In some cases, other combinations of the foregoingare used, for example, contacting the biomass with a protease and apolysaccharide-degrading enzymes in combination with heating the biomassto a temperature of at least 30 degrees Celsius.

In various embodiments in accordance with the present invention,treating the lysate comprises treating with more than about 5% v/v of anorganic solvent to the lysate. In other cases, the treating stepcomprises treating with more than about 6% v/v of an organic solvent tothe lysate, or with more than about 7% v/v of an organic solvent to thelysate. In still other cases, the v/v of the organic solvent to thelysate is between greater than about 5% and greater than about 25%. Inone embodiment, treatment of the lysate can be facilitated by agitatingthe lysate.

In embodiments of the present invention the organic solvent can comprisean oil. The oil can be selected from the group consisting of oil fromsoy, rapeseed, canola, palm, palm kernel, coconut, corn, wastevegetable, Chinese tallow, olive, sunflower, cotton seed, chicken fat,beef tallow, porcine tallow, microalgae, macroalgae, Cuphea, flax,peanut, choice white grease, lard, Camelina sativa, mustard seed cashewnut, oats, lupine, kenaf, calendula, hemp, coffee, linseed, hazelnut,euphorbia, pumpkin seed, coriander, camellia, sesame, safflower, rice,tung oil tree, cocoa, copra, pium poppy, castor beans, pecan, jojoba,jatropha, macadamia, Brazil nuts, avocado, a fossil oil, or a distillatefraction thereof. In some cases the oil is soy oil. In some cases theoil is palm oil. In other cases the oil is coconut oil. In still othercases the oil is canola oil. In yet other cases the oil is jatropha oil.In one embodiment, the time sufficient to allow the lipid from themicroorganism to become solubilized in the organic solvent is between0.1 and 30 minutes.

In various embodiments in accordance with the present invention,separating the lysate comprises centrifugation of the treated lysate,whereby the lysate is separated into a light layer comprising thelipid:organic solvent composition and a heavy layer comprising theaqueous composition and, optionally, an emulsified composition and/orcell pellet composition. In some cases, separating the lysate comprisessettling of the treated lysate, whereby the lysate is separated into alight layer comprising the lipid:organic solvent composition and a heavylayer comprising the aqueous composition and, optionally, an emulsifiedcomposition and/or cell pellet composition. Separation of the lysate caninclude reducing the temperature of the mixture below 25° C., below 10°C., or to a temperature at or below 0° C. In some aspects of theinvention, the layers further comprise a lipid:aqueous emulsion layerand/or a cell pellet.

In some cases, methods of the invention further comprise removing thelipid:organic solvent composition from the other layer(s). In oneembodiment, removal is performed without substantially separating thelipid from the organic solvent.

In some methods of the invention, the microorganism is produced in aculturing process and then optionally stored for a period of timebetween termination of the culturing process and undertaking additionalsteps to lyse the microorganism. In some cases, lysing themicroorganisms in the biomass produces a lysate comprising alipid:aqueous emulsion. In some cases, the microorganism is stored forat least one hour between termination of the culturing process andundertaking additional steps to lyse the cultured microorganism. In somecases, the microorganism is stored for at least twenty-four hoursbetween termination of the culturing process and undertaking additionalsteps to lyse the cultured microorganism. In some cases, themicroorganism is stored for at least forty-eight hours betweentermination of the culturing process and undertaking additional steps tolyse the cultured microorganism. In other cases, the fermentation brothis stored. In some cases, the fermentation broth is concentrated, forexample, by centrifugation or filtration, and the cells are resuspendedin an aqueous media before storage. In some cases the aqueous media isdeionized or distilled water.

In some embodiments of the present invention, microorganisms (biomass)prepared in a culture process are optionally stored at a temperaturebelow 15 degrees Celsius between termination of the culturing processand undertaking additional steps to lyse the cultured microorganism. Insome cases, the biomass is stored at a temperature below 5 degreesCelsius between termination of the culturing process and undertakingadditional steps to lyse the cultured microorganisms. In some cases, themicroorganism is stored at a temperature above 30 degrees Celsiusbetween termination of the culturing process and undertaking additionalsteps to lyse the cultured microorganism. In some cases, themicroorganism is stored at a temperature above 40 degrees Celsiusbetween termination of the culturing process and undertaking additionalsteps to lyse the cultured microorganism. In some cases, themicroorganism is subjected to agitation during storage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts two lipid profiles for pure oils prepared according tothe method of the present invention. Fatty acids C10:1, C10, C12, C18:3,C14, C18:2, C166, C18:1, and C18:0 of pure Chlorella oil (light-shadedbars) and coconut oil (dark-shaded bars) are shown. The majority of pureChlorella oil comprises C18:1 fatty acids (59%), while the majority ofpure coconut oil comprises C12 fatty acids (54%). Details are describedin Example 6.

FIG. 2 depicts the ratio of C18:1 to C12 fatty acids and C18:1 to C14fatty acids in pure algal/coconut oil mixture. Details are described inExample 6.

FIG. 3 depicts results of chemical/heat treatment of Chlorellaprotothecoides. Details are described in Example 7.

FIG. 4 depicts an image of oil layer recovered from a frozen, heattreated emulsion. Details are described in Example 7 (Enzyme Treatment#1).

FIG. 5 depicts a result of a thin layer chromatography (TLC) analysis ofcontrol, basic, and acid generated oil samples. Lane 1, Reference @ 100μg/each (FAME/TAG/FFA); Lane 2, No chemical treatment; Lane 3, treatmentwith 120 mN H2SO4; Lane 4, treatment with 160 mN KOH; Lane 5, nochemical treatment; Lane 6, treatment with 120 mN H2SO4; Lane 7,treatment with 160mN KOH. FAME, fatty acid methyl ester; TAG,triacylglycerides; FFA, free fatty acid; DAG, diacylglycerol(1,3-diolein); MAG, monoacylglycerol (1-monoolein) Details are describedin Example 7.

FIG. 6 depicts an image of layers recovered from enzyme treated (lefttube) versus untreated (right tube) cell culture material. Details aredescribed in Example 7 (Enzyme Treatment #2).

FIG. 7 shows maps of the cassettes used in Prototheca transformations,as described in Example 9.

FIG. 8 shows the results of Southern blot analysis on threetransformants of UTEX strain 1435, as described in Example 9.

FIG. 9 shows a schematic of the codon optimized and non-codon optimizedsuc2 (yeast sucrose invertase (yInv)) transgene construct. The relevantrestriction cloning sites are indicated and arrows indicate thedirection of transcription.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs.

The following references provide one of skill with a general definitionof many of the terms used in this invention: Singleton et al.,Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); TheCambridge Dictionary of Science and Technology (Walker ed., 1988); TheGlossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag(1991); and Hale & Marham, The Harper Collins Dictionary of Biology(1991). As used herein, the following terms have the meanings ascribedto them unless specified otherwise.

As used with reference to a nucleic acid, the phrase “active inmicroalgae” refers to a nucleic acid that is functional in microalgae.For example, a promoter that has been used to drive an antibioticresistance gene to impart antibiotic resistance to a transgenicmicroalgae is active in microalgae. Examples of promoters active inmicroalgae are promoters endogenous to certain algae species andpromoters found in plant viruses.

As used herein, an “acyl carrier protein” or “ACP” is a protein whichbinds a growing acyl chain during fatty acid synthesis as a thiol esterat the distal thiol of the 4′-phosphopantetheine moiety and comprises acomponent of the fatty acid synthase complex.

An “acyl-CoA molecule” or “acyl-CoA” is a molecule comprising an acylmoiety covalently attached to coenzyme A through a thiol ester linkageat the distal thiol of the 4′-phosphopantetheine moiety of coenzyme A.

As used herein, the term “alkyl” refers to a straight or branched chainhydrocarbon radical, and can include di- and multivalent radicals,having the number of carbon atoms designated (i.e. C1-C10 means one toten carbons). Examples of saturated hydrocarbon radicals include groupssuch as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, homologs and isomers of, for example, n-pentyl, n-hexyl,n-heptyl, n-octyl, and the like.

As used herein, the term “alkenyl” refers to an unsaturated alkyl groupone having one or more double bonds. Examples of alkenyl groups includevinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl and 3-(1,4-pentadienyl), and the higher homologs andisomers.

As used herein, the term “alkynyl” refers to an unsaturated alkyl groupone having one or more triple bonds. Examples of alkynyl groups includeethynyl (acetylenyl), 1-propynyl, 1- and 2-butynyl, and the higherhomologs and isomers.

As used herein, the phrase “aqueous or emulsified composition” refers tomicrobial biomass that contains lipid.

As used herein, the phrase “area percent” refers to the area of peaksobserved using FAME GC/FID detection methods in which every fatty acidin the sample is converted into a fatty acid methyl ester (FAME) priorto detection. For examples, a separate peak is observed for a fatty acidof 14 carbon atoms with no unsaturation (C14:0) compared to any otherfatty acid such as a C14:1. The peak area for each class of FAME isdirectly proportional to its percent composition in the mixture and iscalculated based on the sum of all peaks present in the sample (i.e.,[area under specific peak/total area of all measured peaks]×100). Whenreferring to lipid profiles of oils and cells of the invention, “atleast 4% C8-C14,” for example, means that at least 4% of the total fattyacids in the cell or in the extracted glycerolipid composition have achain length that includes 8, 10, 12, or 14 carbon atoms.

As used herein, the term “aryl” refers to a polyunsaturated, aromatic,hydrocarbon substituent having 5-12 ring members, which can be a singlering or multiple rings (up to three rings) which are fused together orlinked covalently. Non-limiting examples of aryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, and benzyl. Other aryl groups arealso useful in the present invention.

As used herein, the term “axenic” refers to a culture of an organismthat is free from contamination by other living organisms.

As used herein, the term “base” refers to any compound whose pKa isgreater than that of water.

As used herein, the term “biodiesel” refers to a fatty acid esterproduced from transesterification of a lipid.

As used herein, the term “biomass” refers to material produced by growthand/or propagation of cells. Biomass may contain cells and/orintracellular contents as well as extracellular material. Extracellularmaterial includes, but is not limited to, compounds secreted by a cell.

As used herein, the term “bioreactor” refers to an enclosure or partialenclosure in which cells, e.g., microorganisms, are cultured, optionallyin suspension.

As used herein, the term “catalyst” refers to an agent, such as amolecule or macromolecular complex, capable of facilitating or promotinga chemical reaction of a reactant to a product without becoming a partof the product. A catalyst thus increases the rate of a reaction, afterwhich, the catalyst may act on another reactant to form the product. Acatalyst generally lowers the overall activation energy required for thereaction such that it proceeds more quickly or at a lower temperature.Thus a reaction equilibrium may be more quickly attained. Examples ofcatalysts include enzymes, which are biological catalysts, and heat,which is a non-biological catalyst.

As used herein, the term “cellulosic material” means the products ofdigestion of cellulose, such as glucose, xylose, arabinose,disaccharides, oligosaccharides, lignin, furfurals, and other molecules.

As used herein, the term “co-culture” and variants thereof, such as“co-cultivate,” refer to the presence of two or more types of cells inthe same bioreactor. The two or more types of cells may both bemicroorganisms, such as microalgae, or may be a microalgal cell culturedwith a different cell type. The culture conditions may be those thatfoster growth and/or propagation of the two or more cell types or thosethat facilitate growth and/or proliferation of one, or a subset, of thetwo or more cells while maintaining cellular growth for the remainder.

As used herein, the term “cofactor” refers to any molecule, other thanthe substrate, that is required for an enzyme to carry out its enzymaticactivity.

A “constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions.

As used herein, the term “cultivated” and variants thereof, refer to theintentional fostering of growth (increases in cell size, cellularcontents, and/or cellular activity) and/or propagation (increases incell numbers via mitosis) of one or more cells by use of intendedculture conditions. The combination of both growth and propagation maybe termed proliferation. The one or more cells may be those of amicroorganism, such as microalgae. Examples of intended conditionsinclude the use of a defined medium (with known characteristics such aspH, ionic strength, and carbon source), specified temperature, oxygentension, carbon dioxide levels, and growth in a bioreactor. The termdoes not refer to the growth or propagation of microorganisms in natureor otherwise without direct human intervention, such as natural growthof an organism that ultimately becomes fossilized to produce geologicalcrude oil.

As used herein, the term “cycloalkyl” refers to a saturated cyclichydrocarbon having 3 to 15 carbons, and 1 to 3 rings that can be fusedor linked covalently. Cycloalkyl groups useful in the present inventioninclude, but are not limited to, cyclopentyl, cyclohexyl, cycloheptyland cyclooctyl. Bicycloalkyl groups useful in the present inventioninclude, but are not limited to, [3.3.0]bicyclooctanyl,[2.2.2]bicyclooctanyl, [4.3.0]bicyclononane, [4.4.0]bicyclodecane(decalin), spiro[3.4]octanyl, spiro[2.5]octanyl, and so forth.

As used herein, the term “cycloalkenyl” refers to an unsaturated cyclichydrocarbon having 3 to 15 carbons, and 1 to 3 rings that can be fusedor linked covalently. Cycloalkenyl groups useful in the presentinvention include, but are not limited to, cyclopentenyl, cyclohexenyl,cycloheptenyl and cyclooctenyl. Bicycloalkenyl groups are also useful inthe present invention.

As used herein, the term “cytolysis” refers to the lysis of cells in ahypotonic environment. Cytolysis is caused by excessive osmosis, ormovement of water, towards the inside of a cell (hyperhydration). Thecell membrane cannot withstand the osmotic pressure of the water inside,and so it explodes.

As used herein, the term “exogenous gene” refers to a nucleic acidtransformed into a cell. A transformed cell may be referred to as arecombinant cell, into which additional exogenous gene(s) may beintroduced. The exogenous gene may be from a different species (and soheterologous), or from the same species (and so homologous) relative tothe cell being transformed. In the case of a homologous gene, itoccupies a different location in the genome of the cell relative to theendogenous copy of the gene. The exogenous gene may be present in morethan one copy in the cell. The exogenous gene may be maintained in acell as an insertion into the genome or as an episomal molecule.

As used herein, the term “exogenously provided” in the context ofculturing a cell, refers to a molecule provided to a culture media of acell culture.

As used herein, the terms “expression vector” or “expression construct”refer to a nucleic acid construct, generated recombinantly orsynthetically, with a series of specified nucleic acid elements thatpermit transcription of a particular nucleic acid in a host cell. Theexpression vector can be part of a plasmid, virus, or nucleic acidfragment. Typically, the expression vector includes a nucleic acid to betranscribed operably linked to a promoter.

As used herein, a “fatty acyl-ACP thioesterase” is an enzyme thatcatalyzes the cleavage of a fatty acid from an acyl carrier protein(ACP) during lipid synthesis.

As used herein, a “fatty acyl-CoA/aldehyde reductase” is an enzyme thatcatalyzes the reduction of an acyl-CoA molecule to a primary alcohol.

As used herein, a “fatty acyl-CoA reductase” is an enzyme that catalyzesthe reduction of an acyl-CoA molecule to an aldehyde.

As used herein, a “fatty aldehyde decarbonylase” is an enzyme thatcatalyzes the conversion of a fatty aldehyde to an alkane.

As used herein, a “fatty aldehyde reductase” is an enzyme that catalyzesthe reduction of an aldehyde to a primary alcohol.

As used herein, the term “fixed carbon source” means molecule(s)containing carbon, preferably organic, that are present at ambienttemperature and pressure in solid or liquid form.

As used herein, the term “fungus,” means heterotrophic organismscharacterized by a chitinous cell wall from the kingdom of fungi.

As used herein, the term “heteroaryl” refers to a polyunsaturated,aromatic, hydrocarbon substituent having 5-12 ring members, which can bea single ring or multiple rings (up to three rings) which are fusedtogether or linked covalently, and which has at least one heteroatom inthe ring, such as N, O, or S. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofheteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Additional heteroaryl groups useful in thepresent invention include pyridyl N-oxide, tetrazolyl, benzofuranyl,benzothienyl, indazolyl, or any of the radicals substituted, especiallymono- or di-substituted.

As used herein, the term “heteroatom” means any atom that is not carbonor hydrogen. Examples of heteroatoms include magnesium, calcium,potassium, sodium, sulfur, phosphorus, iron and copper.

As used herein, the term “heterocycloalkyl” refers to a saturated cyclichydrocarbon having 3 to 15 ring members, and 1 to 3 rings that can befused or linked covalently, and which has at least one heteroatom in thering, such as N, O, or S. Additionally, a heteroatom can occupy theposition at which the heterocycle is attached to the remainder of themolecule. Examples of heterocycloalkyl include 1(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

As used herein, in the context of biomass, the term “homogenate” meansbiomass that has been physically disrupted.

As used herein, “hydrocarbon” refers to: (a) a molecule containing onlyhydrogen and carbon atoms wherein the carbon atoms are covalently linkedto form a linear, branched, cyclic, or partially cyclic backbone towhich the hydrogen atoms are attached; or (b) a molecule that onlyprimarily contains hydrogen and carbon atoms and that can be convertedto contain only hydrogen and carbon atoms by one to four chemicalreactions. Nonlimiting examples of the latter include hydrocarbonscontaining an oxygen atom between one carbon and one hydrogen atom toform an alcohol molecule, as well as aldehydes containing a singleoxygen atom. Methods for the reduction of alcohols to hydrocarbonscontaining only carbon and hydrogen atoms are well known. Anotherexample of a hydrocarbon is an ester, in which an organic group replacesa hydrogen atom (or more than one) in an oxygen acid. The molecularstructure of hydrocarbon compounds varies from the simplest, in the formof methane (CH4), which is a constituent of natural gas, to the veryheavy and very complex, such as some molecules such as asphaltenes foundin crude oil, petroleum, and bitumens. Hydrocarbons may be in gaseous,liquid, or solid form, or any combination of these forms, and may haveone or more double or triple bonds between adjacent carbon atoms in thebackbone. Accordingly, the term includes linear, branched, cyclic, orpartially cyclic alkanes, alkenes, lipids, and paraffin. Examplesinclude propane, butane, pentane, hexane, octane, squalene andcarotenoids.

As used herein, the term “hydrocarbon modification enzyme” refers to anenzyme that alters the covalent structure of a hydrocarbon. Examples ofhydrocarbon modification enzymes include a lipase, a fatty acyl-ACPthioesterase, a fatty acyl-CoA/aldehyde reductase, a fatty acyl-CoAreductase, a fatty aldehyde reductase, and a fatty aldehydedecarbonylase.

As used herein, the term “hydrogen:carbon ratio” refers to the ratio ofhydrogen atoms to carbon atoms in a molecule on an atom-to-atom basis.The ratio may be used to refer to the number of carbon and hydrogenatoms in a hydrocarbon molecule. For example, the hydrocarbon with thehighest ratio is methane CH₄ (4:1).

As used herein, the term “hydrophobic fraction” refers to a portion, orfraction, of a material that is more soluble in a hydrophobic phase incomparison to an aqueous phase. A hydrophobic fraction is substantiallyinsoluble in water and usually non-polar.

As used herein, the phrase “increase lipid yield” refers to an increasein the productivity of a microbial culture by, for example, increasingdry weight of cells per liter of culture, increasing the percentage ofcells that constitute lipid, or increasing the overall amount of lipidper liter of culture volume per unit time.

An “inducible promoter” is one that mediates transcription of anoperably linked gene in response to a particular stimulus.

As used herein, the term “in situ” means “in place” or “in its originalposition.” For example, a culture may contain a first microalgaesecreting a catalyst and a second microorganism secreting a substrate,wherein the first and second cell types produce the components necessaryfor a particular chemical reaction to occur in situ in the co-culturewithout requiring further separation or processing of the materials.

As used herein, the term “isomers” refers to compounds of the presentinvention that possess asymmetric carbon atoms (optical centers) ordouble bonds. The racemates, diastereomers, geometric isomers andindividual isomers are all intended to be encompassed within the scopeof the present invention.

As used herein, the phrase “limiting concentration of a nutrient” refersto a concentration in a culture that limits the propagation of acultured organism. A “non-limiting concentration of a nutrient” is aconcentration that supports maximal propagation during a given cultureperiod. Thus, the number of cells produced during a given culture periodis lower in the presence of a limiting concentration of a nutrient thanwhen the nutrient is non-limiting. A nutrient is said to be “in excess”in a culture, when the nutrient is present at a concentration greaterthan that which supports maximal propagation.

As used herein, a “lipase” is an enzyme that catalyzes the hydrolysis ofester bonds in water-insoluble, lipid substrates. Lipases catalyze thehydrolysis of lipids into glycerols and fatty acids.

“Lipids” are a class of molecules that are soluble in nonpolar solvents(such as ether and chloroform) and are relatively or completelyinsoluble in water. Lipid molecules have these properties because theyconsist largely of long hydrocarbon tails that are hydrophobic innature. Examples of lipids include fatty acids (saturated andunsaturated); glycerides or glycerolipids (such as monoglycerides,diglycerides, triglyceries or neutral fats and phosophoglycerides orglycerophospholipids); nonglycerides (sphingolipids, sterol lipidsincluding cholesterol and steroid hormones, prenol lipids includingterpenoids, fatty alcohols, waxes and polyketides); and complex lipidderivatives (sugar-linked lipids, or glycolipids, and protein-linkedlipids). “Fats” are a subgroup of lipids called “triacylglycerides”.

As used herein, the phrase “lipid:organic solvent composition” refers toa mixture of lipid and organic solvent.

As used herein, a “lipid pathway enzyme” is any enzyme that plays a rolein lipid metabolism, i.e., either lipid synthesis, modification, ordegradation. This term encompasses proteins that chemically modifylipids, as well as carrier proteins.

As used herein, the term “lysate” refers to a solution containing thecontents of lysed cells.

As used herein, the term “lysis” refers to the breakage of the plasmamembrane and optionally the cell wall of a biological organismsufficient to release at least some intracellular content, often bymechanical, viral or osmotic mechanisms that compromise its integrity.

As used herein, the term “lysing” refers to disrupting the cellularmembrane and optionally the cell wall of a biological organism or cellsufficient to release at least some intracellular content.

As used herein, the term “microalgae” means a microbial organism that iseither (a) eukaryotic and contains a chloroplast or chloroplast remnant,or (b) is a cyanobacteria. Microalgae include obligate photoautotrophs,which cannot metabolize a fixed carbon source as energy, as well asheterotrophs, which can live solely off of a fixed carbon source.Microalgae can refer to unicellular organisms that separate from sistercells shortly after cell division, such as Chlamydomonas, and can alsorefer to microbes such as, for example, Volvox, which is a simplemulticellular photosynthetic microbe of two distinct cell types.“Microalgae” can also refer to cells such as Chlorella and Dunaliella.“Microalgae” also includes other microbial photosynthetic organisms thatexhibit cell-cell adhesion, such as Agmenellum, Anabaena, andPyrobotrys, as well as organisms that contain chloroplast-likestructures that are no longer capable of performing photosynthesis, suchas microalgae of the genus Prototheca and some dinoflagellates.Microalgae” also includes obligate heterotrophic micoorganisms that havelost the ability to perform photosynthesis, such as certaindinoflagellate species.

The terms “microorganism” and “microbe” are used interchangeably hereinto refer to microscopic unicellular organisms.

As used herein, the term “oil” means a hydrophobic, lipophilic, nonpolarcarbon-containing substance including but not limited togeologically-derived crude oil, distillate fractions ofgeologically-derived crude oil, vegetable oil, algal oil, and microbiallipids.

As used herein, the term “oleaginous yeast,” means yeast that canaccumulate more than 10% of its dry cell weight as lipid. Oleaginousyeast includes organisms such as Yarrowia lipolytica, as well asengineered strains of yeast such as Saccharomyces cerevisiae that havebeen engineered to accumulate more than 10% of the dry cell weight aslipid.

As used herein, the terms “operably linked,” “in operable linkage,” orgrammatical equivalents thereof refer to a functional linkage betweentwo sequences, such a control sequence (typically a promoter) and thelinked sequence. A promoter is in operable linkage with an exogenousgene if it can mediate transcription of the gene.

As used herein, the term “organic solvent” refers to a carbon-containingmaterial that dissolves a solid, liquid, or gaseous solute, resulting ina solution.

As used herein, the term “osmotic shock” refers to the rupture ofbacterial, algal, or other cells in a solution following a suddenreduction in osmotic pressure. Osmotic shock is sometimes induced torelease cellular components of such cells into a solution.

As used herein, the term “photobioreactor” refers to a container, atleast part of which is at least partially transparent or partially open,thereby allowing light to pass through, in which, e.g., one or moremicroalgae cells are cultured. Photobioreactors may be closed, as in theinstance of a polyethylene bag or Erlenmeyer flask, or may be open tothe environment, as in the instance of an outdoor pond.

As used herein, the term “polysaccharide” (also called “glycan”) refersto carbohydrate made up of monosaccharides joined together by glycosidiclinkages. Cellulose is an example of a polysaccharide that makes upcertain plant cell walls. Cellulose can be depolymerized by enzymes toyield monosaccharides such as xylose and glucose, as well as largerdisaccharides and oligosaccharides.

As used herein, the term “polysaccharide-degrading enzyme” refers to anyenzyme capable of catalyzing the hydrolysis, or depolymerization, of anypolysaccharide. For example, cellulase catalyzes the hydrolysis ofcellulose.

As used herein, the term “port,” in the context of a bioreactor, refersto an opening in the bioreactor that allows influx or efflux ofmaterials such as gases, liquids, and cells. Ports are usually connectedto tubing leading from the photobioreactor.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription.

As used herein, the term “recombinant” when used with reference, e.g.,to a cell, or nucleic acid, protein, or vector, indicates that the cell,nucleic acid, protein or vector, has been modified by the introductionof a heterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid as depicted above.

As used herein, the term “renewable diesel” refers to a mixture ofalkanes (such as C10:0, C12:0, C14:0, C16:0 and C18:0) produced throughhydrogenation and deoxygenation of lipids. Renewable diesel alsoincludes diesel fuel derived from biomass as defined in Section45K(c)(3), using the process of thermal depolymerization (EPAct 2005).

As used herein, the term “sonication” refers to a process of disruptingbiologic materials, such as a cell, by use of sound wave energy.

As used herein, the term “wastewater” refers to a watery waste whichtypically contains washing water, laundry waste, faeces, urine, andother liquid or semi-liquid wastes. It includes some forms of municipalwaste as well as secondarily treated sewage.

II. General

U.S. Patent application Nos. 60/941,581; 60/959,174; 60/968,291; and61/024,069 are hereby incorporated by reference in their entirety forall purposes.

The invention generally relates to the production of hydrocarboncompositions, such as a lipid, in microorganisms. In particular, theinvention provides methods for extracting, recovering, isolating andobtaining a lipid from a microorganism and compositions comprising thelipid. The invention also discloses methods for producing hydrocarboncompositions for use as biodiesel, renewable diesel, jet fuel, and forproducing a lipid surfactant having a carbon chain length of C12 andC14.

The invention is premised in part on the insight that certainmicroorganisms can be used to produce hydrocarbon compositionseconomically and in large quantities for use in the transportation fueland petrochemical industry among other applications. Suitablemicroorganisms include microalgae, oleaginous yeast, and fungi. Apreferred genus of microalgae for use in the invention is thelipid-producing microalgae Chlorella. Acidic transesterification oflipids yields long-chain fatty acid esters useful as biodiesel. Otherenzymatic processes can be tailored to yield fatty acids, aldehydes,alcohols, and alkanes. The present application describes methods forgenetic modification of multiple species and strains of microorganisms,including Chlorella and similar microbes to provide organisms havingcharacteristics that facilitate the production of lipids suitable forconversion into biodiesel or other hydrocarbon compounds. The presentapplication also describes methods of cultivating microalgae forincreased productivity and increased lipid yield.

Microorganisms useful in the invention produce lipids or hydrocarbonssuitable for biodiesel production or as feedstock for industrialapplications. Suitable hydrocarbons for biodiesel production includetriacylglycerides (TAGs) containing long-chain fatty acid molecules.Suitable hydrocarbons for industrial applications, such as surfactantmanufacturing, include fatty acids, aldehydes, alcohols, and alkanes. Insome embodiments, suitable fatty acids, or the corresponding primaryalcohols, aldehydes, or alkanes, generated by the methods describedherein, contain at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, at least 27, at least28, at least 29, at least 30, at least 31, at least 32, at least 33, atleast 34, or at least 35 carbon atoms or more. Preferred long-chainfatty acids for biodiesel generally contain at least 14 carbon atoms ormore.

Preferred fatty acids, or the corresponding primary alcohols, aldehydes,and alkanes, for industrial applications contain at least 8 carbon atomsor more. In certain embodiments, the above fatty acids, as well as theother corresponding hydrocarbon molecules, are saturated (with nocarbon-carbon double or triple bonds); mono unsaturated (single doublebond); poly unsaturated (two or more double bonds); are linear (notcyclic); and/or have little or no branching in their structures.

Triacylglycerols containing carbon chain lengths in the C8 to C22 rangeare preferred. Preferred for surfactants are C10-C14. Preferred forbiodiesel or renewable diesel are C16 to C18. Preferred for jet fuel areC8-C10. Preferred for nutrition are C22 polyunsaturated fatty acids(such as DHA) and carotenoids (such as astaxanthin).

III. Microorganisms Useful for Producing Lipids

Any species of organism that produces suitable lipid or hydrocarbon canbe used, although microorganisms that naturally produce high levels ofsuitable lipid or hydrocarbon are preferred. Production of hydrocarbonsby microorganisms is reviewed by Metzger et al. Appl MicrobiolBiotechnol (2005) 66: 486-496 and A Look Back at the U.S. Department ofEnergy's Aquatic Species Program: Biodiesel from Algae,NREL/TP-580-24190, John Sheehan, Terri Dunahay, John Benemann and PaulRoessler (1998).

Considerations affecting the selection of a microorganism for use in theinvention include, in addition to production of suitable hydrocarbonsfor biodiesel or for industrial applications: (1) high lipid content asa percentage of cell weight; (2) ease of growth; (3) ease of geneticengineering; and (3) ease of processing. In particular embodiments, thewild-type or genetically engineered microorganism yields cells that areat least: about 40%, about 45%, about 50%, about 55%, about 60%, about65%, or about 70% or more lipid. Preferred organisms growheterotrophically (on sugar in the absence of light) or can beengineered to do so using, for example, methods disclosed incommonly-owned U.S. Patent Application Nos. 60/837,839 and 60/968,291,which are incorporated herein by reference in their entireties. The easeof transformation and availability of selectable markers and promoters,constitutive and/or inducible, that are functional in the microorganismaffect the ease of genetic engineering. Processing considerations caninclude, for example, the availability of effective means for lysing thecells.

A. Algae

In a preferred embodiment of the present invention, a microorganismproducing a lipid or a microorganism from which a lipid can beextracted, recovered, or obtained, is an algae.

Examples of algae that can be used to practice the present inventioninclude, but are not limited to the following algae listed in Table 1.

TABLE 1 Examples of algae. Achnanthes orientalis, Agmenellum, Amphiprorahyaline, Amphora coffeiformis, Amphora coffeiformis linea, Amphoracoffeiformis punctata, Amphora coffeiformis taylori, Amphoracoffeiformis tenuis, Amphora delicatissima, Amphora delicatissimacapitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmusfalcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii,Botryococcus sudeticus, Carteria, Chaetoceros gracilis, Chaetocerosmuelleri, Chaetoceros muelleri subsalsum, Chaetoceros sp., Chlorellaanitrata, Chlorella Antarctica, Chlorella aureoviridis, Chlorellacandida, Chlorella capsulate, Chlorella desiccate, Chlorellaellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var.vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorellainfusionum var. actophila, Chlorella infusionum var. auxenophila,Chlorella kessleri, Chlorella lobophora (strain SAG 37.88), Chlorellaluteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorellaluteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima,Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorellaphotophila, Chlorella pringsheimii, Chlorella protothecoides (includingany of UTEX strains 1806, 411, 264, 256, 255, 250, 249, 31, 29, 25, andCCAP strains 211/17 and 211/8d), Chlorella protothecoides var.acidicola, Chlorella regularis, Chlorella regularis var. minima,Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorellasaccharophila, Chlorella saccharophila var. ellipsoidea, Chlorellasalina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp.,Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii,Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia,Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis,Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f.tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorellaxanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorellavulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium,Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Cryptomonas sp.,Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Dunaliellasp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate,Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliellapeircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola,Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta,Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena,Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp.,Gloeothamnion sp., Hymenomonas sp., Isochrysis aff. galbana, Isochrysisgalbana, Lepocinclis, Micractinium, Micractinium (UTEX LB 2614),Monoraphidium minutum, Monoraphidium sp., Nannochloris sp.,Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata,Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa,Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp.,Nitschia communis, Nitzschia alexandrina, Nitzschia communis, Nitzschiadissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschiainconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschiapusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis,Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva,Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoriasp., Oscillatoria subbrevis, Pascheria acidophila, Pavlova sp., Phagus,Phormidium, Platymonas sp., Pleurochrysis carterae, Pleurochrysisdentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora,Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii,Pyramimonas sp., Pyrobotrys, Sarcinoid chrysophyte, Scenedesmus armatus,Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp.,Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosiraweissflogii, and Viridiella fridericiana

1. Chlorella and Prototheca

In a preferred embodiment of the present invention, the microorganismproducing a lipid or a microorganism from which a lipid can beextracted, recovered, or obtained, is of the genus Chlorella,preferably, Chlorella protothecoides.

Chlorella is a genus of single-celled green algae, belonging to thephylum Chlorophyta. It is spherical in shape, about 2 to 10 μm indiameter, and is without flagella. Some species of Chlorella arenaturally heterotrophic.

Chlorella, particularly Chlorella protothecoides, is a preferredmicroorganism for use in the invention because of its high composition(at least about 55% by weight) of lipid, particularly long-chain lipidsuitable for biodiesel. In addition, this microalgae growsheterotrophically, can be genetically engineered as demonstrated in theExamples herein, and can be lysed by Chlorella virus.

In a preferred embodiment of the present invention, the microorganismused for expression of a transgene is of the genus Chlorella,preferably, Chlorella protothecoides. Examples of expression oftransgenes in, e.g., Chlorella, can be found in the literature (see forexample Current Microbiology Vol. 35 (1997), pp. 356-362; Sheng Wu GongCheng Xue Bao. 2000 July; 16(4):443-6; Current Microbiology Vol. 38(1999), pp. 335-341; Appl Microbiol Biotechnol (2006) 72: 197-205;Marine Biotechnology 4, 63-73, 2002; Current Genetics 39:5, 365-370(2001); Plant Cell Reports 18:9, 778-780, (1999); Biologia Plantarium42(2): 209-216, (1999); Plant Pathol. J 21(1): 13-20, (2005)). Also seeExamples herein. Other lipid-producing microbes can be engineered aswell, including prokaryotic microbes (see Kalscheuer et al., AppliedMicrobiology and Biotechnology, volume 52, number 4/October, 1999).

In another preferred embodiment of the present invention, themicroorganism producing a lipid or a microorganism from which a lipidcan be extracted, recovered, or obtained, is of the genus Prototheca,preferably, Prototheca moriformis. Species of the genus Prototheca aresuited for the production of lipid because they can produce high levelsof lipids, particularly lipids suitable for fuel and chemicalproduction. The lipid produced by Prototheca has fatty acid chains ofshorter chain length and a higher degree of saturation than the lipidproduced by most microalgae. Moreover, Prototheca lipid is generallyfree of pigment (low to undetectable levels of chlorophyll and certaincarotenoids) and in any event contains much less pigment than lipid fromother microalgae. Illustrative Prototheca strains for use in the methodsof the invention include Prototheca wickerhamii, Prototheca stagnora(including UTEX 327), Prototheca portoricensis, Prototheca moriformis(including UTEX strains 1441, 1435), and Prototheca zopfii. Species ofthe genus Prototheca are obligate heterotrophs.

2. Identification of Microalgal Species

Species of microalgae, including Chlorella and Prototheca, for use inthe invention can be identified by amplification of certain targetregions of the genome. For example, identification of a specificChlorella species can be achieved through amplification and sequencingof nuclear and/or chloroplast DNA using primers and methodologydescribed in Wu et al., Identification of Chlorella spp. isolates usingany region of the genome. Examples include ribosomal DNA sequences, Bot.Bull. Acad. Sin. (2001) 42:115-121. Well established methods ofphylogenetic analysis, such as amplification and sequencing of ribosomalinternal transcribed spacer (ITS1 and ITS2 rDNA), 18S rRNA, and otherconserved genomic regions can be used by those skilled in the art toidentify species of not only Chlorella or Prototheca, but otherhydrocarbon and lipid producing organisms capable of using the methodsdisclosed herein. For examples of methods of identification andclassification of algae also see for example Genetics, 2005 August;170(4):1601-10 and RNA, 2005 April; 11(4):361-4.

Genomic DNA comparison can be used to identify species of microalgae tobe used in the present invention. Regions of conserved genomic DNA, suchas but not limited to DNA encoding 23S rRNA, can be amplied frommicroalgal species and compared to consensus sequences in order toscreen for microalgal species that are taxonomically related to thepreferred microalgae used in the present invention. Examples of such DNAsequence comparison for species within the Chlorella genus are shownbelow.

Genomic DNA comparison can also be useful to identify microalgal speciesthat have been misidentified in a strain collection. Often a straincollection will identify species of microalgae based on phenotypic andmorphological characteristics. The use of these characteristics may leadto miscategorization of the species or the genus of a microalgae. Theuse of genomic DNA comparison can be a better method of categorizingmicroalgae species based on their phylogenetic relationship. Specificexamples of using genotyping data to establish phyogenetic relationshipsof possibly misidentified microalgal strains are described below in theExamples.

In some cases, microalgae that are preferred for use in the presentinvention have genomic DNA sequences encoding for 23S rRNA that have atleast 99%, at least 98%, at least 97%, at least 96%, at least 95%, atleast 94%, at least 93%, at least 92%, at least 91%, at least 90%, atleast 89%, at least 88%, at least 87%, or at least 86% nucleotideidentity to at least one of the sequences listed in SEQ ID NOs: 7-31. Inother cases, microalgae that are preferred for use in the presentinvention have genomic DNA sequences encoding for 23S rRNA that have atleast 85%, at least 80%, at least 75% at least 70% at least 65% or atleast 60% nucleotide identity to at least one of the sequences listed inSEQ ID NOs. 7-31, 32, 33, 34, 35, 36, 37, 38, and 39.

B. Oleaginous Yeast

In a preferred embodiment of the present invention, a microorganismproducing a lipid or a microorganism from which a lipid can beextracted, recovered, or obtained, is an oleaginous yeast.

Examples of oleaginous yeast that can be used to practice the presentinvention include, but are not limited to the following oleaginous yeastlisted in Table 2.

TABLE 2 Examples of oleaginous yeast. Candida apicola, Candida sp.,Cryptococcus curvatus, Cryptococcus terricolus, Debaromyces hansenii,Endomycopsis vernalis, Geotrichum carabidarum, Geotrichum cucujoidarum,Geotrichum histeridarum, Geotrichum silvicola, Geotrichum vulgare,Hyphopichia burtonii, Lipomyces lipofer, Lypomyces orentalis, Lipomycesstarkeyi, Lipomyces tetrasporous, Pichia mexicana, Rodosporidiumsphaerocarpum, Rhodosporidium toruloides Rhodotorula aurantiaca,Rhodotorula dairenensis, Rhodotorula diffluens, Rhodotorula glutinus,Rhodotorula glutinis var. glutinis, Rhodotorula gracilis, Rhodotorulagraminis Rhodotorula minuta, Rhodotorula mucilaginosa, Rhodotorulamucilaginosa var. mucilaginosa, Rhodotorula terpenoidalis, Rhodotorulatoruloides, Sporobolomyces alborubescens, Starmerella bombicola,Torulaspora delbruekii, Torulaspora pretoriensis, Trichosporon behrend,Trichosporon brassicae , Trichosporon domesticum, Trichosporonlaibachii, Trichosporon loubieri, Trichosporon loubieri var. loubieri,Trichosporon montevideense, Trichosporon pullulans, Trichosporon sp.,Wickerhamomyces Canadensis, Yarrowia lipolytica, and Zygoascus meyerae.

In a preferred embodiment of the present invention, the microorganismused for expression of a transgene is an oleaginous yeast. Examples ofexpression of transgenes in oleaginous yeast (e.g., Yarrowia lipolytica)can be found in the literature (see, for example, Bordes et al., JMicrobiol Methods, June 27 (2007)).

C. Other Fungi

In a preferred embodiment of the present invention, a microorganismproducing a lipid or a microorganism from which a lipid can beextracted, recovered, or obtained, is a fungus.

Examples of fungi that can be used to practice the present inventioninclude, but are not limited to the following fungi listed in Table 3.

TABLE 3 Examples of non-yeast fungi. Mortierella, Mortierrla vinacea,Mortierella alpine, Pythium debaryanum, Mucor circinelloides,Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum,Hensenulo, Chaetomium, Cladosporium, Malbranchea, Rhizopus, and Pythium

In a preferred embodiment of the present invention, the microorganismused for expression of a transgene is a fungus. Examples of expressionof transgenes in fungi (e.g., Mortierella alpine, Mucor circinelloides,and Aspergillus ochraceus) can also be found in the literature (see, forexample, Microbiology, July; 153(Pt. 7):2013-25 (2007); Mol GenetGenomics, June; 271(5):595-602 (2004); Curr Genet, March; 21(3):215-23(1992); Current Microbiology, 30(2):83-86 (1995); Sakuradani, NISRResearch Grant, “Studies of Metabolic Engineering of UsefulLipid-producing Microorganisms” (2004); and PCT/JP2004/012021).

D. Bacteria

In a preferred embodiment of the present invention, a microorganismproducing a lipid or a microorganism from which a lipid can beextracted, recovered, or obtained, is a bacterium.

Examples of expression of exogenous genes in bacteria, such as E. coli,are well known; see for example Molecular Cloning: A Laboratory Manual,Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press).

IV. Microbe Engineering

In certain embodiments of the present invention, it is desirous togenetically modify a microorganism. Thus, the present applicationdescribes genetically engineering strains of microalgae, oleaginousyeast, bacteria, or fungi with one or more exogenous genes to producevarious hydrocarbon compounds.

Promoters, cDNAs, and 3′UTRs, as well as other elements of expressionvectors, can be generated through cloning techniques using fragmentsisolated from native sources (see for example Molecular Cloning: ALaboratory Manual, Sambrook et al. (3d edition, 2001, Cold Spring HarborPress; and U.S. Pat. No. 4,683,202). Alternatively, elements can begenerated synthetically using known methods (see for example Gene, 1995Oct. 16; 164(1):49-53).

A. Codon-Optimization for Expression

DNA encoding a polypeptide to be expressed in a microorganism, e.g., alipase and selectable marker are preferably codon-optimized cDNA.Methods of recoding genes for expression in microalgae are described inU.S. Pat. No. 7,135,290. Additional information for codon optimizationis available, e.g., at the codon usage database of GenBank. Asnon-limiting examples, codon usage in Prototheca species, Dunaliellasalina, and Chlorella protothecoides are shown in Tables 4, 5, and 6,respectively.

TABLE 4 Codon usage in Prototheca species. Ala GCG 345 (0.36) Asn AAT 8(0.04) GCA 66 (0.07) AAC 201 (0.96) GCT 101 (0.11) Pro CCG 161 (0.29)GCC 442 (0.46) CCA 49 (0.09) Cys TGT 12 (0.10) CCT 71 (0.13) TGC 105(0.90) CCC 267 (0.49) Asp GAT 43 (0.12) Gln CAG 226 (0.82) GAC 316(0.88) CAA 48 (0.18) Glu GAG 377 (0.96) Arg AGG 33 (0.06) GAA 14 (0.04)AGA 14 (0.02) Phe TTT 89 (0.29) CGG 102 (0.18) TTC 216 (0.71) CGA 49(0.08) Gly GGG 92 (0.12) CGT 51 (0.09) GGA 56 (0.07) CGC 331 (0.57) GGT76 (0.10) Ser AGT 16 (0.03) GGC 559 (0.71) AGC 123 (0.22) His CAT 42(0.21) TCG 152 (0.28) CAC 154 (0.79) TCA 31 (0.06) Ile ATA 4 (0.01) TCT55 (0.10) ATT 30 (0.08) TCC 173 (0.31) ATC 338 (0.91) Thr ACG 184 (0.38)Lys AAG 284 (0.98) ACA 24 (0.05) AAA 7 (0.02) ACT 21 (0.05) Leu TTG 26(0.04) ACC 249 (0.52) TTA 3 (0.00) Val GTG 308 (0.50) CTG 447 (0.61) GTA9 (0.01) CTA 20 (0.03) GTT 35 (0.06) CTT 45 (0.06) GTC 262 (0.43) CTC190 (0.26) Trp TGG 107 (1.00) Met ATG 191 (1.00) Tyr TAT 10 (0.05) TAC180 (0.95) Stop TGA/TAG/TAA

TABLE 5 Preferred codon usage in Dunaliella salina. TTC (Phe) TAC (Tyr)TGC (Cys) TAA (Stop) TGG (Trp) CCC (Pro) CAC (His) CGC (Arg) CTG (Leu)CAG (Gln) ATC (Ile) ACC (Thr) AAC (Asn) AGC (Ser) ATG (Met) AAG (Lys)GCC (Ala) GAC (Asp) GGC (Gly) GTG (Val) GAG (Glu)

TABLE 6 Preferred codon usage in Chlorella protothecoides. TTC (Phe) TAC(Tyr) TGC (Cys) TGA (Stop) TGG (Trp) CCC (Pro) CAC (His) CGC (Arg) CTG(Leu) CAG (Gln) ATC (Ile) ACC (Thr) GAC (Asp) TCC (Ser) ATG (Met) AAG(Lys) GCC (Ala) AAC (Asn) GGC (Gly) GTG (Val) GAG (Glu)

B. Promoters

Many promoters are active in microalgae, including promoters that areendogenous to the algae being transformed, as well as promoters that arenot endogenous to the algae being transformed (i.e., promoters fromother algae, promoters from higher plants, and promoters from plantviruses or algae viruses). Exogenous and/or endogenous promoters thatare active in microalgae, and antibiotic resistance genes functional inmicroalgae are described by e.g., Curr Microbiol. 1997 December;35(6):356-62 (Chlorella vulgaris); Mar Biotechnol (NY). 2002 January;4(1):63-73 (Chlorella ellipsoidea); Mol Gen Genet. 1996 Oct. 16;252(5):572-9 (Phaeodactylum tricornutum); Plant Mol. Biol. 1996 April;31(1):1-12 (Volvox carteri); Proc Natl Acad Sci USA. 1994 Nov. 22;91(24):11562-6 (Volvox carteri); Falciatore A, Casotti R, Leblanc C,Abrescia C, Bowler C, PMID: 10383998, 1999 May; 1(3):239-251 (Laboratoryof Molecular Plant Biology, Stazione Zoologica, Villa Comunale, 1-80121Naples, Italy) (Phaeodactylum tricornutum and Thalassiosiraweissflogii); Plant Physiol. 2002 May; 129(1):7-12. (Porphyridium sp.);Proc Natl Acad Sci USA. 2003 Jan. 21; 100(2):438-42. (Chlamydomonasreinhardtii); Proc Natl Acad Sci USA. 1990 February; 87(3):1228-32.(Chlamydomonas reinhardtii); Nucleic Acids Res. 1992 Jun. 25;20(12):2959-65; Mar Biotechnol (NY). 2002 January; 4(1):63-73(Chlorella); Biochem Mol Biol Int. 1995 August; 36(5):1025-35(Chlamydomonas reinhardtii); J Microbiol. 2005 August; 43(4):361-5(Dunaliella); Yi Chuan Xue Bao. 2005 April; 32(4):424-33 (Dunaliella);Mar Biotechnol (NY). 1999 May; 1(3):239-251. (Thalassiosira andPhaedactylum); Koksharova, Appl Microbiol Biotechnol 2002 February;58(2):123-37 (various species); Mol Genet Genomics 2004 February;271(1):50-9 (Thermosynechococcus elongates); J. Bacteriol. (2000), 182,211-215; FEMS Microbiol Lett. 2003 Apr. 25; 221(2):155-9; Plant Physiol.1994 June; 105(2):635-41; Plant Mol. Biol. 1995 December; 29(5):897-907(Synechococcus PCC 7942); Mar Pollut Bull. 2002; 45(1-12):163-7(Anabaena PCC 7120); Proc Natl Acad Sci USA. 1984 March; 81(5):1561-5(Anabaena (various strains)); Proc Natl Acad Sci USA. 2001 Mar. 27;98(7):4243-8 (Synechocystis); Wirth, Mol Gen Genet. 1989 March;216(1):175-7 (various species); Mol Microbiol, 2002 June; 44(6):1517-31;Plasmid, 1993 September; 30(2):90-105 (Fremyella diplosiphon); Hall etal. (1993) Gene 124: 75-81 (Chlamydomonas reinhardtii); Gruber et al.(1991). Current Micro. 22: 15-20; Jarvis et al. (1991) Current Genet.19: 317-322 (Chlorella); for additional promoters see also Table 1 fromU.S. Pat. No. 6,027,900).

The promoter used to express an exogenous gene can be the promoternaturally linked to that gene or can be a heterologous gene. Somepromoters are active in more than one species of microalgae. Otherpromoters are species-specific. Preferred promoters include promoterssuch as RBCS2 from Chlamydomonas reinhardtii and viral promoters, suchas cauliflower mosaic virus (CMV) and chlorella virus, which have beenshown to be active in multiple species of microalgae (see for examplePlant Cell Rep. 2005 March; 23(10-11):727-35; J Microbiol. 2005 August;43(4):361-5; Mar Biotechnol (NY). 2002 January; 4(1):63-73). In otherembodiments, the Botryococcus malate dehydrogenase promoter, such anucleic acid comprising any part of SEQ ID NO:3, or the Chlamydomonasreinhardtii RBCS2 promoter (SEQ ID NO:4) can be used. Optionally, atleast 10, 20, 30, 40, 50, or 60 nucleotides or more of these sequencescontaining a promoter are used. Preferred promoters endogenous tospecies of the genus Chlorella are SEQ ID NOs: 1 and 2.

Preferred promoters useful for expression of exogenous genes inChlorella are listed in the sequence listing of this application, suchas the promoter of the Chlorella HUP1 gene (SEQ ID NO:1) and theChlorella ellipsoidea nitrate reductase promoter (SEQ ID NO:2).Chlorella virus promoters can also be used to express genes inChlorella, such as SEQ ID NOs: 1-7 of U.S. Pat. No. 6,395,965.Additional promoters active in Chlorella can be found, for example, inBiochem Biophys Res Commun, 1994 Oct. 14; 204(1):187-94; Plant Mol Biol,1994 October; 26(1):85-93; Virology, 2004 Aug. 15; 326(1):150-9; andVirology, 2004 Jan. 5; 318(1):214-23.

C. Selectable Markers

Any of a wide variety of selectable markers can be employed in atransgene construct useful for transforming a microorganism, e.g.,Chlorella. Examples of suitable selectable markers include the nitratereductase gene, the hygromycin phosphotransferase gene (HPT), theneomycin phosphotransferase gene, and the ble gene, which confersresistance to phleomycin. Methods of determining sensitivity ofmicroalgae to antibiotics are well known. For example, Mol Gen Genet,1996 Oct. 16; 252(5):572-9.

More specifically, Dawson et al. (1997), Current Microbiology 35:356-362(incorporated by reference herein in its entirety), described the use ofthe nitrate reductase (NR) gene from Chlorella vulgaris as a selectablemarker for NR-deficient Chlorella sorokiniana mutants. Kim et al.(2002), Mar. Biotechnol. 4:63-73 (incorporated by reference herein inits entirety), disclosed the use of the HPT gene as a selectable markerfor transforming Chorella ellipsoidea. Huang et al. (2007), Appl.Microbiol. Biotechnol. 72: 197-205 (incorporated by reference herein inits entirety), reported on the use of Sh ble as a selectable marker forChlorella sp. DT.

D. Inducible Expression

The present invention also provides for the use of an inducible promoterto express a gene of interest. In particular, the use of an induciblepromoter to express a lipase gene permits production of the lipase aftergrowth of the microorganism when conditions have been adjusted, ifnecessary, to enhance transesterification, for example, after disruptionof the cells, reduction of the water content of the reaction mixture,and/or addition sufficient alcohol to drive conversion of TAGs to fattyacid esters.

Inducible promoters useful in the invention include those that mediatetranscription of an operably linked gene in response to a stimulus, suchas an exogenously provided small molecule (e.g., glucose, as in SEQ IDNO:1), temperature (heat or cold), light, etc. Suitable promoters canactivate transcription of an essentially silent gene or upregulate,preferably substantially, transcription of an operably linked gene thatis transcribed at a low level. In the latter case, the level oftranscription of the gene of interest, e.g., the lipase gene, preferablydoes not significantly interfere with the growth of the microorganism inwhich it is expressed. Expression of a transgene in Chlorella can beperformed under inducible conditions, e.g., using promoters such as thepromoter for the Chlorella hexose transporter gene (SEQ ID NO:1). Thispromoter is strongly activated by the presence of glucose in the culturemedia.

E. Expression of Two or More Exogenous Genes

Further, a genetically engineered microorganism, such as a microalgae,may comprise and express two or more exogenous genes, such as, forexample, a lipase and a lytic gene, e.g., one encoding apolysaccharide-degrading enzyme. One or both genes can be expressedusing an inducible promoter, which allows the relative timing ofexpression of these genes to be controlled to enhance the lipid yieldand conversion to fatty acid esters. Expression of the two or moreexogenous genes may be under control of the same inducible promoter orunder control of a different inducible promoters. In the lattersituation, expression of a first exogenous gene can be induced for afirst period of time (during which expression of a second exogenous genemay or may not be induced) and expression of a second exogenous gene canbe induced for a second period of time (during which expression of afirst exogenous gene may or may not be induced). Provided herein arevectors and methods for engineering lipid-producing microbes tometabolize sucrose, which is an advantageous trait because it allows theengineered cells to convert sugar cane feedstocks into lipidsappropriate for biodiesel production.

Also provided herein are genetically engineered strains of microbes(e.g., microalgae, oleaginous yeast, bacteria, or fungi) that expresstwo or more exogenous genes, such as, for example, a fatty acyl-ACPthioesterase and a fatty acyl-CoA/aldehyde reductase, the combinedaction of which yields an alcohol product. Further provided are othercombinations of exogenous genes, including without limitation, a fattyacyl-ACP thioesterase and a fatty acyl-CoA reductase to generatealdehydes. In addition, this application provides for the combination ofa fatty acyl-ACP thioesterase, a fatty acyl-CoA reductase, and a fattyaldehyde decarbonylase to generate alkanes. One or more of the exogenousgenes can be expressed using an inducible promoter.

Examples of further modifications suitable for use in the presentinvention are described in co-pending, commonly owned Application No.60/837,839, which is incorporated herein by reference. This applicationdiscloses genetically engineering strains of microalgae to express twoor more exogenous genes, one encoding a transporter of a fixed carbonsource (such as sucrose) and a second encoding a sucrose invertaseenzyme. The resulting fermentable organisms produce hydrocarbons atlower manufacturing cost than what has been obtainable by previouslyknown methods of biological hydrocarbon production. This co-pendingapplication also teaches that the insertion of the two exogenous genesdescribed above can be combined with the disruption of polysaccharidebiosynthesis through directed and/or random mutagenesis, which steersever greater carbon flux into hydrocarbon production. Individually andin combination, trophic conversion, engineering to alter hydrocarbonproduction and treatment with exogenous enzymes alter the hydrocarboncomposition produced by a microorganism. The alteration can be a changein the amount of hydrocarbons produced, the amount of one or morehydrocarbon species produced relative to other hydrocarbons, and/or thetypes of hydrocarbon species produced in the microorganism. For example,microalgae can be engineered to produce a higher amount and/orpercentage of TAGs.

F. Compartmentalized Expression

The present invention also provides for compartmentalized expression agene of interest. In particular, it can be advantageous, in particularembodiments, to target expression of a lipase gene, to one or morecellular compartments, where it is sequestered from the majority ofcellular lipids until initiation of the transesterification reaction.Preferred organelles for targeting are chloroplasts, mitochondria, andendoplasmic reticulum.

1. Expression in Chloroplasts

In a preferred embodiment of the present invention, the expression of apolypeptide in a microorganism is targeted to chloroplasts. Methods fortargeting expression of a heterologous gene to the chloroplast are knownand can be employed in the present invention. Methods for targetingforeign gene products into chloroplasts are described in Shrier et al.,EMBO J. (1985) 4:25 32. See also Tomai et al., Gen. Biol. Chem. (1988)263:15104 15109 and U.S. Pat. No. 4,940,835 for the use of transitpeptides for translocating nuclear gene products into the chloroplast.Methods for directing the transport of proteins to the chloroplast arealso reviewed in Kenauf TIBTECH (1987) 5:40 47. Chloroplast targetingsequences endogenous to Chlorella are known, such as genes in theChlorella nuclear genome that encode proteins that are targeted to thechloroplast; see for example GenBank Accession numbers AY646197 andAF499684.

Wageningen UR-Plant Research International sells an IMPACTVECTOR1.4vector, which uses the secretion signal of the Chrysanthemum morifoliumsmall subunit protein to deliver a heterologous protein into thechloroplast stroma (cytoplasmic) environment, shuttling across a doublemembrane system. The protein is fused to the first 11 amino acids of themature rubisco protein in order to allow proper processing of the signalpeptide (Wong et al., Plant Molecular Biology 20: 81-93 (1992)). Thesignal peptide contains a natural intron from the RbcS gene.

In another approach, the chloroplast genome is genetically engineered toexpress the heterologous protein. Stable transformation of chloroplastsof Chlamydomonas reinhardtii (a green alga) using bombardment ofrecipient cells with high-velocity tungsten microprojectiles coated withforeign DNA has been described. See, for example, Boynton et al.,Science (1988) 240: 1534 1538; Blowers et al. Plant Cell (1989) 1:123132 and Debuchy et al., EMBO J. (1989) δ: 2803 2809. The transformationtechnique, using tungsten microprojectiles, is described by Klein etal., Nature (London) (1987) 7:70 73. Other methods of chloroplasttransformation for both plants and microalgae are known. See for exampleU.S. Pat. Nos. 5,693,507; 6,680,426; Plant Physiol. 2002 May;129(1):7-12; and Plant Biotechnol J. 2007 May; 5(3):402-12.

As described in U.S. Pat. No. 6,320,101 (issued Nov. 20, 2001 to Kaplanet al.; which is incorporated herein by reference), cells can bechemically treated so as to reduce the number of chloroplasts per cellto about one. Then, the heterologous nucleic acid can be introduced intothe cells via particle bombardment with the aim of introducing at leastone heterologous nucleic acid molecule into the chloroplasts. Theheterologous nucleic acid is selected such that it is integratable intothe chloroplast's genome via homologous recombination which is readilyeffected by enzymes inherent to the chloroplast. To this end, theheterologous nucleic acid includes, in addition to a gene of interest,at least one nucleic acid sequence that is derived from thechloroplast's genome. In addition, the heterologous nucleic acidtypically includes a selectable marker. Further details relating to thistechnique are found in U.S. Pat. Nos. 4,945,050 and 5,693,507, which areincorporated herein by reference. A polypeptide can thus be produced bythe protein expression system of the chloroplast.

U.S. Pat. No. 7,135,620 (issued Nov. 14, 2006 to Daniell et al.;incorporated herein by reference) describes chloroplast expressionvectors and related methods. Expression cassettes are DNA constructsincluding a coding sequence and appropriate control sequences to providefor proper expression of the coding sequence in the chloroplast. Typicalexpression cassettes include the following components: the 5′untranslated region from a microorganism gene or chloroplast gene suchas psbA which will provide for transcription and translation of a DNAsequence encoding a polypeptide of interest in the chloroplast; a DNAsequence encoding a polypeptide of interest; and a translational andtranscriptional termination region, such as a 3′ inverted repeat regionof a chloroplast gene that can stabilize RNA of introduced genes,thereby enhancing foreign gene expression. The cassette can optionallyinclude an antibiotic resistance gene.

Typically, the expression cassette is flanked by convenient restrictionsites for insertion into an appropriate genome. The expression cassettecan be flanked by DNA sequences from chloroplast DNA to facilitatestable integration of the expression cassette into the chloroplastgenome, particularly by homologous recombination. Alternatively, theexpression cassette may remain unintegrated, in which case, theexpression cassette typically includes a chloroplast origin ofreplication, which is capable of providing for replication of theheterologous DNA in the chloroplast.

The expression cassette generally includes a promoter region from a genecapable of expression in the chloroplast. The promoter region mayinclude promoters obtainable from chloroplast genes, such as the psbAgene from spinach or pea, or the rbcL and atpB promoter region frommaize and rRNA promoters. Examples of promoters are described inHanley-Bowdoin and Chua, TIBS (1987) 12:67 70; Mullet et al., PlantMolec Biol. (1985) 4: 39 54; Hanley-Bowdoin (1986) PhD. Dissertation,the Rockefeller University; Krebbers et al., Nucleic Acids Res. (1982)10: 4985 5002; Zurawaki et al., Nucleic Acids Res. (1981) 9:3251 3270;and Zurawski et al., Proc. Natl. Acad. Sci. U.S.A. (1982) 79: 7699 7703.Other promoters can be identified and the relative strength of promotersso identified evaluated, by placing a promoter of interest 5′ to apromoterless marker gene and observing its effectiveness relative totranscription obtained from, for example, the promoter from the psbAgene, a relatively strong chloroplast promoter. The efficiency ofheterologous gene expression additionally can be enhanced by any of avariety of techniques. These include the use of multiple promotersinserted in tandem 5′ to the heterologous gene, for example a doublepsbA promoter, the addition of enhancer sequences and the like.

Numerous promoters active in the Chlorella chloroplast can be used forexpression of exogenous genes in the Chlorella chloroplast, such asthose found in GenBank accession number NC_(—)001865 (Chlorella vulgarischloroplast, complete genome).

Where it is desired to provide for inducible expression of theheterologous gene, an inducible promoter and/or a 5′ untranslated regioncontaining sequences which provide for regulation at the level oftranscription and/or translation (at the 3′ end) may be included in theexpression cassette. For example, the 5′ untranslated region can be froma gene wherein expression is regulatable by light. Similarly, 3′inverted repeat regions could be used to stabilize RNA of heterologousgenes. Inducible genes may be identified by enhanced expression inresponse to a particular stimulus of interest and low or absentexpression in the absence of the stimulus. For example, alight-inducible gene can be identified where enhanced expression occursduring irradiation with light, while substantially reduced expression orno expression occurs in low or no light. Light regulated promoters fromgreen microalgae are known (see for example Mol Genet Genomics, 2005December; 274(6):625-36).

The termination region which is employed will be primarily one ofconvenience, since the termination region appears to be relativelyinterchangeable among chloroplasts and bacteria. The termination regionmay be native to the transcriptional initiation region, may be native tothe DNA sequence of interest, or may be obtainable from another source.See, for example, Chen and Orozco, Nucleic Acids Res. (1988) 16:8411.

The expression cassettes may be transformed into a plant cell ofinterest by any of a number of methods. These methods include, forexample, biolistic methods (See, for example, Sanford, Trends InBiotech. (1988) 6:299 302, U.S. Pat. No. 4,945,050; electroporation(Fromm et al., Proc. Natl. Acad. Sci. (USA) (1985) 82:5824 5828); use ofa laser beam, microinjection or any other method capable of introducingDNA into a chloroplast.

Additional descriptions of chloroplast expression vectors suitable foruse in microorganisms such as microalgae are found in U.S. Pat. Nos.7,081,567 (issued Jul. 25, 2006 to Xue et al.); 6,680,426 (issued Jan.20, 2004 to Daniell et al.); and 5,693,507 (issued Dec. 2, 1997 toDaniell et al.).

Proteins expressed in the nuclear genome of Chlorella can be targeted tothe chloroplast using chloroplast targeting signals. Chloroplasttargeting sequences endogenous to Chlorella are known, such as genes inthe Chlorella nuclear genome that encode proteins that are targeted tothe chloroplast; see for example GenBank Accession numbers AY646197 andAF499684. Proteins can also be expressed in the Chlorella chloroplast byinsertion of genes directly into the chloroplast genome. Chloroplasttransformation typically occurs through homologous recombination, andcan be performed if chloroplast genome sequences are known for creationof targeting vectors (see for example the complete genome sequence of aChlorella chloroplast; Genbank accession number NC_(—)001865). Seeprevious sections herein for details of chloroplast transformation.

2. Expression in Mitochondria

In another preferred embodiment of the present invention, the expressionof a polypeptide in a microorganism is targeted to mitochondria. Methodsfor targeting foreign gene products into mitochondria (Boutry et al.,Nature (London) (1987) 328:340 342) have been described, including ingreen microalgae (see for example Mol Gen Genet., 1993 January;236(2-3):235-44).

For example, an expression vector encoding a suitable secretion signalcan target a heterologous protein to the mitochondria. An exemplaryexpression vector for mitochondria targeting is the IMPACTVECTOR1.5vector, from Wageningen UR-Plant Research International, which uses theyeast CoxIV secretion signal. This expression vector was shown todeliver proteins in the mitochondrial matrix. The protein is fused tothe first 4 amino acids of the yeast CoxIV protein in order to allowproper processing of the signal peptide (Kohler et al., Plant J. 11:613-621 (1997)). Other mitochondrial targeting sequences are known,including those functional in green microalgae. For example, see FEBSLett. 1990 Jan. 29; 260(2):165-8; and J. Biol. Chem. 2002 Feb. 22;277(8):6051-8.

Proteins expressed in the nuclear genome of Chlorella can be targeted tothe mitochondria using mitochondrial targeting signals. Details ofmitochondrial protein targeting and transformation are provided herein.

3. Expression in Endoplasmic Reticulum

In another preferred embodiment of the present invention, the expressionof a polypeptide in a microorganism is targeted to the endoplasmicreticulum (ER). The inclusion of an appropriate retention or sortingsignal in an expression vector ensure that proteins are retained in theendoplasmic reticulum (ER) and do not go downstream into Golgi. Forexample, the IMPACTVECTOR1.3 vector, from Wageningen UR-Plant ResearchInternational, includes the well known KDEL retention or sorting signal.With this vector, ER retention has a practical advantage in that it hasbeen reported to improve expression levels 5-fold or more. The mainreason for this appears to be that the ER contains lower concentrationsand/or different proteases responsible for post-translationaldegradation of expressed proteins than are present in the cytoplasm. ERretention signals functional in green microalgae are known. For example,see Proc. Nat.l Acad. Sci. USA. 2005 Apr. 26; 102(17):6225-30.

G. Transformation

Cells can be transformed by any suitable technique including, e.g.,biolistics, electroporation (see Maruyama et al., (2004) BiotechnologyTechniques 8:821-826), glass bead transformation and silicon carbidewhisker transformation. Another method that can be used involves formingprotoplasts and using CaCl₂ and polyethylene glycol (PEG) to introducerecombinant DNA into microalgal cells (see Kim et al., (2002), Mar.Biotechnol. 4:63-73, which reports the use of this method for thetransformation of Chlorella ellipsoidea). Co-transformation ofmicroalgae can be used to introduce two distinct vector molecules into acell simultaneously (see for example, Protist (2004) December;155(4):381-93).

Biolistic methods (see for example, Sanford, Trends in Biotech. (1988)6:299-302, U.S. Pat. No. 4,945,050; electroporation (Fromm et al., PNAS(1985) 82:5824-5828), use of a laser beam, microinjection, or any othermethod capable of introducing DNA into a microalgae can also be used fortransformation.

H. Lipid Pathway Engineering

In certain embodiments of the present invention, it is preferred tofurther modify a microorganism, such as a microalgae, for example, toprovide desired growth characteristics and/or to enhance the amountand/or quality of lipids produced. For example, microalgae can beengineered to increase carbon flux into the lipid pathway and/or modifythe lipid pathway to beneficially alter the proportions or properties oflipid produced by the cells. The pathway is further, or alternatively,modified to alter the properties and/or proportions of varioushydrocarbon molecules produced through enzymatic processing of lipids.

1. Alteration of Properties or Portions of Lipids or HydrocarbonsProduced

In some embodiments of the present invention, it can be desirable toalter characteristics, such as lipid yield per unit volume and/or perunit time, carbon chain length (e.g., for biodiesel production or forindustrial applications requiring hydrocarbon feedstock), reducing thenumber of double or triple bonds, optionally to zero, removing oreliminating rings and cyclic structures, and increasing thehydrogen:carbon ratio of a particular species of lipid or of apopulation of distinct lipid. In addition, microalgae that produceappropriate hydrocarbons can also be engineered to have even moredesirable hydrocarbon outputs. Examples of such microalgae includespecies of the genus Chlorella.

a) Regulation of Enzymes Controlling Branch Points in Fatty AcidSynthesis

In particular embodiments of the present invention, one or more keyenzymes that control branch points in metabolism to fatty acid synthesisis up-regulated or down-regulated to improve lipid production.Up-regulation is achieved, for example, by transforming cells withexpression constructs in which a gene encoding the enzyme of interest isexpressed, e.g., using a strong promoter and/or enhancer elements thatincrease transcription. Such expression constructs can include aselectable marker such that the transformants can be subjected toselection, which can result in amplification of the construct and anincrease in the expression level of the encoded enzyme. Examples ofenzymes suitable for up-regulation according to the methods of theinvention include pyruvate dehydrogenase, which plays a role inconverting pyruvate to acetyl-CoA (examples, some from microalgae,include GenBank accession numbers NP_(—)415392; AAA53047; Q1XDM1; andCAF05587). Up-regulation of pyruvate dehydrogenase can increaseproduction of acetyl-CoA, and thereby increase fatty acid synthesis.Acetyl-CoA carboxylase catalyzes the initial step in fatty acidsynthesis. Accordingly, in certain embodiments of the present invention,this enzyme is up-regulated to increase production of fatty acids(examples, some from microalgae, include GenBank accession numbersBAA94752; AAA75528; AAA81471; YP_(—)537052; YP_(—)536879; NP_(—)045833;and BAA57908). In another embodiment, fatty acid production is increasedby up-regulation of acyl carrier protein (ACP), which carries thegrowing acyl chains during fatty acid synthesis (examples, some frommicroalgae, include GenBank accession numbers A0T0F8; P51280;NP_(—)849041; YP_(—)874433). Glycerol-3-phosphate acyltransferasecatalyzes the rate-limiting step of fatty acid synthesis. Up-regulationof this enzyme is desired to increase fatty acid production (examples,some from microalgae, include GenBank accession numbers AAA74319;AAA33122; AAA37647; P44857; and ABO94442). In some embodiments, two ormore of these polypeptides (pyruvate dehydrogenase, Acetyl-CoAcarboxylase, acyl carrier protein (ACP), Glycerol-3-phosphateacyltransferase) are up-regulated. In that case, the two or more genesencoding the respective polypeptides may reside on a single expressionconstruct or, alternatively, on two or more expression constructs. Thepreceding proteins are candidates for expression in microalgae,including species of the genus Chlorella and/or Prototheca.

Down-regulation of an enzyme of interest can achieved using, e.g.,antisense, catalytic RNA/DNA, RNA interference (RNAi), “knock-out,”“knock-down,” or other mutagenesis techniques. Enzymeexpression/function can also be inhibited using intrabodies. Examples ofenzymes suitable for down-regulation according to the methods of theinvention include citrate synthase, which consumes acetyl-CoA as part ofthe tricarboxylic acid (TCA) cycle. Down-regulation of citrate synthasecan force more acetyl-CoA into the fatty acid synthetic pathway.

b) Modulation of Global Regulators of Fatty Acid Synthetic Genes

Global regulators modulate the expression of the genes of the fatty acidbiosynthetic pathways. Accordingly, one or more global regulators offatty acid synthesis can be up- or down-regulated, as appropriate, toinhibit or enhance, respectively, the expression of a plurality of fattyacid synthetic genes and, ultimately, to increase lipid production.Examples include sterol regulatory element binding proteins (SREBPs),such as SREBP-1a and SREBP-1c (for examples see GenBank accessionnumbers NP_(—)035610 and Q9WTN3). Global regulators can be up- ordown-regulated, for example, as described above with respect toregulation of control point enzymes.

c) Regulation of Hydrocarbon Modification Enzymes

The present application describes genetically engineering strains ofmicroalgae, oleaginous yeast, bacteria, or fungi with one or moreexogenous genes to produce various hydrocarbon compounds. Thus, incertain embodiments of the present invention, the methods of theinvention also comprise transforming cells with one or more genesencoding hydrocarbon modification enzymes, such as, for example, a fattyacyl-ACP thioesterase (see examples in Table 7 with accession numbers),a fatty acyl-CoA/aldehyde reductase (see examples in Table 8 withaccession numbers), a fatty acyl-CoA reductase (see examples in Table 9with accession numbers), a fatty aldehyde decarbonylase (see examples inTable 10 with accession numbers), a fatty aldehyde reductase, or asqualene synthase gene (e.g., see GenBank Accession number AF205791).Stearoyl-ACP desaturase, for example, catalyzes the conversion ofstearoyl-ACP to oleoyl-ACP. Up-regulation of this gene can increase theproportion of monounsaturated fatty acids produced by a cell; whereasdown-regulation can reduce the proportion of monounsaturates. Similarly,the expression of one or more glycerolipid desaturases can be controlledto alter the ratio of unsaturated to saturated fatty acids such as ω-6fatty acid desaturase, ω-3 fatty acid desaturase, or ω-6-oleatedesaturase.

For example, microalgae that would naturally, or through geneticmodification, produce high levels of lipids can be engineered (orfurther engineered) to express an exogenous fatty acyl-ACP thioesterase,which can facilitate the cleavage of fatty acids from the acyl carrierprotein (ACP) during lipid synthesis. These fatty acids can be recoveredor, through further enzymatic processing within the cell, yield otherhydrocarbon compounds. Optionally, the fatty acyl-ACP thioesterase canbe expressed from a gene operably linked to an inducible promoter,and/or can be expressed in an intracellular compartment.

Thus, in a preferred embodiment of the present invention, thehydrocarbon modification enzyme suitable for use with the microorganismsand methods of the invention is a fatty acyl-ACP thioesterase. Fattyacyl-ACP thioesterases include, without limitation, those listed inTable 7, each of which is hereby incorporated by reference.

TABLE 7 Fatty acyl-ACP thioesterases and GenBank accession numbers.Umbellularia californica fatty acyl-ACP thioesterase (GenBank #AAC49001)Cinnamomum camphora fatty acyl-ACP thioesterase (GenBank #Q39473)Umbellularia californica fatty acyl-ACP thioesterase (GenBank #Q41635)Myristica fragrans fatty acyl-ACP thioesterase (GenBank #AAB71729)Myristica fragrans fatty acyl-ACP thioesterase (GenBank #AAB71730)Elaeis guineensis fatty acyl-ACP thioesterase (GenBank #ABD83939) Elaeisguineensis fatty acyl-ACP thioesterase (GenBank #AAD42220) Populustomentosa fatty acyl-ACP thioesterase (GenBank #ABC47311) Arabidopsisthaliana fatty acyl-ACP thioesterase (GenBank #NP_172327) Arabidopsisthaliana fatty acyl-ACP thioesterase (GenBank #CAA85387) Arabidopsisthaliana fatty acyl-ACP thioesterase (GenBank #CAA85388) Gossypiumhirsutum fatty acyl-ACP thioesterase (GenBank #Q9SQI3) Cuphea lanceolatafatty acyl-ACP thioesterase (GenBank #CAA54060) Cuphea hookeriana fattyacyl-ACP thioesterase (GenBank #AAC72882) Cuphea calophylla subsp.mesostemon fatty acyl-ACP thioesterase (GenBank #ABB71581) Cuphealanceolata fatty acyl-ACP thioesterase (GenBank #CAC19933) Elaeisguineensis fatty acyl-ACP thioesterase (GenBank #AAL15645) Cupheahookeriana fatty acyl-ACP thioesterase (GenBank #Q39513) Gossypiumhirsutum fatty acyl-ACP thioesterase (GenBank #AAD01982) Vitis viniferafatty acyl-ACP thioesterase (GenBank #CAN81819) Garcinia mangostanafatty acyl-ACP thioesterase (GenBank #AAB51525) Brassica juncea fattyacyl-ACP thioesterase (GenBank #ABI18986) Madhuca longifolia fattyacyl-ACP thioesterase (GenBank #AAX51637) Brassica napus fatty acyl-ACPthioesterase (GenBank #ABH11710) Oryza sativa (indica cultivar-group)fatty acyl-ACP thioesterase (GenBank #EAY86877) Oryza sativa (japonicacultivar-group) fatty acyl-ACP thioesterase (GenBank #NP_001068400)Oryza sativa (indica cultivar-group) fatty acyl-ACP thioesterase(GenBank #EAY99617) Cuphea hookeriana fatty acyl-ACP thioesterase(GenBank #AAC49269)

A fatty acyl-ACP thioesterase can be chosen based on its specificity fora growing (during fatty acid synthesis) fatty acid having a particularcarbon chain length. For example, the fatty acyl-ACP thioesterase canhave a specificity for a carbon chain length ranging from 8 to 34 carbonatoms, preferably from 8 to 18 carbon atoms, and more preferably from 10to 14 carbon atoms. A specificity for a fatty acid with 12 carbon atomsis most preferred. A specificity for a fatty acid with 14 carbon atomsis also preferred.

In another preferred embodiment of the present invention, thehydrocarbon modification enzyme suitable for use with the microorganismsand methods of the invention is a fatty acyl-CoA/aldehyde reductase.Fatty acyl-CoA/aldehyde reductases include, without limitation, thoselisted in Table 8.

TABLE 8 Fatty acyl-CoA/aldehyde reductases listed by GenBank accessionnumbers. AAC45217, YP_047869, BAB85476, YP_001086217, YP_580344,YP_001280274, YP_264583, YP_436109, YP_959769, ZP_01736962, ZP_01900335,ZP_01892096, ZP_01103974, ZP_01915077, YP_924106, YP_130411,ZP_01222731, YP_550815, YP_983712, YP_001019688, YP_524762, YP_856798,ZP_01115500, YP_001141848, NP_336047, NP_216059, YP_882409, YP_706156,YP_001136150, YP_952365, ZP_01221833, YP_130076, NP_567936, AAR88762,ABK28586, NP_197634, CAD30694, NP_001063962, BAD46254, NP_001030809,EAZ10132, EAZ43639, EAZ07989, NP_001062488, CAB88537, NP_001052541,CAH66597, CAE02214, CAH66590, CAB88538, EAZ39844, AAZ06658, CAA68190,CAA52019, and BAC84377

In another preferred embodiment of the present invention, thehydrocarbon modification enzyme suitable for use with the microorganismsand methods of the invention is a fatty acyl-CoA reductase. Fattyacyl-CoA reductases include, without limitation, those listed in Table9.

TABLE 9 Fatty acyl-CoA reductases listed by GenBank accession numbers.NP_187805, ABO14927, NP_001049083, CAN83375, NP_191229, EAZ42242,EAZ06453, CAD30696, BAD31814, NP_190040, AAD38039, CAD30692, CAN81280,NP_197642, NP_190041, AAL15288, and NP_190042

In another preferred embodiment of the present invention, thehydrocarbon modification enzyme suitable for use with the microorganismsand methods of the invention is a fatty aldehyde decarbonylase. Fattyaldehyde decarbonylases include, without limitation, those listed inTable 10.

TABLE 10 Fatty aldehyde decarbonylases listed by GenBank accessionnumbers. NP_850932, ABN07985, CAN60676, AAC23640, CAA65199, AAC24373,CAE03390, ABD28319, NP_181306, EAZ31322, CAN63491, EAY94825, EAY86731,CAL55686, XP_001420263, EAZ23849, NP_200588, NP_001063227, CAN83072,AAR90847, and AAR97643

Additional examples of amino acid sequences for hydrocarbon modificationenzymes or nucleic acids encoding them are described in U.S. Pat. Nos.6,610,527, 6,451,576, 6,429,014, 6,342,380, 6,265,639, 6,194,185,6,114,160, 6,083,731, 6,043,072, 5,994,114, 5,891,697, 5,871,988, and6,265,639, and further described in GenBank Accession numbers AAO18435,ZP_(—)00513891, Q38710, AAK60613, AAK60610, AAK60611, NP_(—)113747,CAB75874, AAK60612, AAF20201, BAA11024, AF205791, and CAA03710.

In particular embodiments, microorganisms of the present invention aregenetically engineered to express one or more exogenous genes selectedfrom a fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehyde reductase,a fatty acyl-CoA reductase, a fatty aldehyde reductase, or a fattyaldehyde decarbonylase. Suitable expression methods are described abovewith respect to the expression of a lipase gene, including, among othermethods, inducible expression and compartmentalized expression.

Other suitable enzymes for use with the microrganisms and the methods ofthe invention include those that have at least 70% amino acid identitywith one of the proteins listed in Tables 7-10, and that exhibit thecorresponding desired enzymatic activity (e.g., cleavage of a fatty acidfrom an acyl carrier protein, reduction of an acyl-CoA to an aldehyde oran alcohol, or conversion of an aldehyde to an alkane). In additionalembodiments, the enzymatic activity is present in a sequence that has atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 99% identity with one of theabove described sequences, all of which are hereby incorporated byreference as if fully set forth.

The hydrocarbon modification enzymes described herein are useful in theproduction of various hydrocarbons from a microorganism (e.g., amicroalgae, an oleaginous yeast, or a fungus) or population ofmicroorganisms, whereby a fatty acyl-ACP thioesterase cleaves a fattyacid from an acyl carrier protein (ACP) during lipid synthesis. Throughfurther enzymatic processing, the cleaved fatty acid is then combinedwith a coenzyme to yield an acyl-CoA molecule. This acyl-CoA is thesubstrate for the enzymatic activity of a fatty acyl-CoA reductase toyield an aldehyde, as well as for a fatty acyl-CoA/aldehyde reductase toyield an alcohol. The aldehyde produced by the action of the fattyacyl-CoA reductase identified above is the substrate for furtherenzymatic activity by either a fatty aldehyde reductase to yield analcohol, or a fatty aldehyde decarbonylase to yield an alkane.

The hydrocarbon modification enzymes have a specificity for acting on asubstrate which includes a specific number of carbon atoms. For example,a fatty acyl-ACP thioesterase may have a specificity for cleaving afatty acid having 12 carbon atoms from the ACP. Therefore, in variousembodiments, the microorganism can contain an exogenous gene thatencodes a protein with specificity for catalyzing an enzymatic activity(e.g., cleavage of a fatty acid from an ACP, reduction of an acyl-CoA toan aldehyde or an alcohol, or conversion of an aldehyde to an alkane)with regard to the number of carbon atoms contained in the substrate.The enzymatic specificity can, in various embodiments, be for asubstrate having from 8 to 34 carbon atoms, preferably from 8 to 18carbon atoms, and more preferably from 10 to 14 carbon atoms. The mostpreferred specificity is for a substrate having 12 carbon atoms. In yetanother embodiment, the preferred specificity is for a substrate having14 carbon atoms. In other embodiments the specificity can be for 20 to30 carbon atoms.

By selecting the desired combination of exogenous genes to be expressed,one can tailor the product generated by the microorganism, which maythen be extracted from the aqueous biomass. For example, in certainembodiments, the microorganism contains: (i) an exogenous gene encodinga fatty acyl-ACP thioesterase; and, optionally, (ii) an exogenous geneencoding a fatty acyl-CoA/aldehyde reductase or a fatty acyl-CoAreductase; and,

optionally, (iii) an exogenous gene encoding a fatty aldehyde reductaseor a fatty aldehyde decarbonylase. The microorganism, when cultured asdescribed herein, synthesizes a fatty acid linked to an ACP and thefatty acyl-ACP thioesterase catalyzes the cleavage of the fatty acidfrom the ACP to yield, through further enzymatic processing, a fattyacyl-CoA molecule. When present, the fatty acyl-CoA/aldehyde reducatasecatalyzes the reduction of the acyl-CoA to an alcohol. Similarly, thefatty acyl-CoA reductase, when present, catalyzes the reduction of theacyl CoA to an aldehyde. In those embodiments in which an exogenous geneencoding a fatty acyl-CoA reductase is present and expressed to yield analdehyde product, a fatty aldehyde reductase, encoded by the thirdexogenous gene, catalyzes the reduction of the aldehyde to an alcohol.Similarly, a fatty aldehyde decarbonylase catalyzes the conversion ofthe aldehyde to an alkane, when present in the microrganism.

Genes encoding such enzymes can be obtained from cells already known toexhibit significant lipid production such as Chlorella protothecoides.Genes already known to have a role in lipid production, e.g., a geneencoding an enzyme that saturates double bonds, can be transformedindividually into recipient cells. However, to practice the invention itis not necessary to make a priori ansumptions as to which genes arerequired. A library of DNA containing different genes, such as cDNAsfrom a good lipid-production organism, can be transformed into recipientcells. The cDNA is preferably in operable linkage with a promoter activein microalgae. Different recipient microalgae cells transformed by alibrary receive different genes from the library. Transformants havingimproved lipid production are identified though screening methods knownin the art, such as, for example, HPLC, gas chromatography, and massspectrometry methods of hydrocarbon analysis (for examples of suchanalysis, see Biomass and Bioenergy Vol. 6. No. 4. pp. 269-274 (1994);Experientia 38; 47-49 (1982); and Phytochemistry 65 (2004) 3159-3165).These transformants are then subjected to further transformation withthe original library and/or optionally interbred to generate a furtherround of organisms having improved lipid production. General proceduresfor evolving whole organisms to acquire a desired property are describedin, e.g., U.S. Pat. No. 6,716,631. Such methods entail, e.g.,introducing a library of DNA fragments into a plurality of cells,whereby at least one of the fragments undergoes recombination with asegment in the genome or an episome of the cells to produce modifiedcells. The modified cells are then screened for modified cells that haveevolved toward acquisition of the desired function. Vectors and methodsfor transformation are analogous to those discussed in connection withexpression of lipase genes.

Furthermore, subtractive libraries can be used to identify genes whosetranscription is induced under different conditions, especiallyconditions employed in culturing microorganisms for biodieselproduction, or for the production of hydrocarbons useful as a feedstockfor industrial applications. Subtractive libraries contain nucleotidesequences reflecting the differences between two different samples. Suchlibraries are prepared by procedures that include the steps ofdenaturing and hybridizing populations of polynucleotides (e.g., mRNA,cDNA, amplified sequences) from each sample. Sequences common to bothsamples hybridize and are removed, leaving the sequences that differbetween the samples. In this manner, sequences that are induced underparticular conditions can be identified. This technique can be employed,for example, to identify genes useful for increasing lipid (e.g., fattyacid) production and, in particular, lipid production under any desiredculture conditions. The subtractive hybridization technique can also beemployed to identify promoters, e.g., inducible promoters, useful inexpression constructs according to the invention.

Thus, for example, subtractive libraries can be prepared frommicroorganism cultures grown autotrophically (in the light without afixed carbon source) or heterotrophically (in the dark in the presenceof a fixed carbon source). In particular, heterotrophic genes may beinduced during dark growth in the presence of a fixed carbon source andmay therefore be present in a library generated by subtracting sequencesfrom autotrophic cells from sequences from dark heterotrophic cells.Subtractive libraries can also be prepared from cultures to which aparticular carbon substrate, such as glucose, has been added to identifygenes that play a role in metabolizing the substrate. Subtractivelibraries prepared from cultures grown in the presence of excess versuslimited nitrogen can be used to identify genes that control celldivision as opposed to hydrocarbon accumulation production. Thepreparation of a subtractive library from a culture to which lipids(e.g., fatty acids) have been added can help identify genes whoseoverexpression increases fatty acid production. More specifically, theaddition of fatty acids to a culture of cells that can use the addedfatty acids will lead to the down-regulation of fatty acid syntheticgenes to down-regulate fatty acid production. The overexpression of oneor more such genes will have the opposite effect.

2. Increased Carbon Flux Into Lipid Pathway

Some microalgae produce significant quantities of non-lipid metabolites,such as, for example, polysaccharides. Because polysaccharidebiosynthesis can use a significant proportion of the total metabolicenergy available to cells, mutagenesis of lipid-producing cells followedby screening for reduced or eliminated polysaccharide productiongenerates novel strains that are capable of producing higher yields oflipids.

The phenol: sulfuric acid assay detects carbohydrates (see Hellebust,Handbook of Phycological Methods, Cambridge University Press, 1978; andCuesta G., et al., J. Microbiol. Methods, 2003 January; 52(1):69-73).The 1,6 dimethylmethylene blue assay detects anionic polysaccharides.(see for example Braz. J. Med. Biol. Res. 1999 May; 32(5):545-50; Clin.Chem. 1986 November; 32(11):2073-6).

Polysaccharides can also be analyzed through methods such as HPLC, sizeexclusion chromatography, and anion exchange chromatography (see forexample Prosky L, Asp N, Schweizer T F, DeVries J W & Furda I (1988)Determination of insoluble, soluble and total dietary fiber in food andfood products: Interlaboratory study. Journal of the Association ofOfficial Analytical Chemists 71, 1017±1023; Int J Biol Macromol. 2003November; 33(1-3):9-18). Polysaccharides can also be detected using gelelectrophoresis (see for example, Anal Biochem. 2003 Oct. 15;321(2):174-82; Anal Biochem. 2002 Jan. 1; 300(1):53-68).

V. Methods of Culturing Microorganisms

A. Bioreactor

Microorganisms are cultured both for purposes of conducting geneticmanipulations and for subsequent production of hydrocarbons (e.g.,lipids, fatty acids, aldehydes, alcohols, and alkanes). The former typeof culture is conducted on a small scale and initially, at least, underconditions in which the starting microorganism can grow. For example, ifthe starting microorganism is a photoautotroph the initial culture isconducted in the presence of light. The culture conditions can bechanged if the microorganism is evolved or engineered to growindependently of light. Culture for purposes of hydrocarbon productionis preferentially conducted on a large scale (e.g., 10,000 L, 40,000 L,100,000 L or larger bioreactors) in a bioreactor. Microorganisms (e.g.,microalgae) of the invention are typically cultured in the methods ofthe invention in liquid media within a bioreactor, typically in theabsence of light (heterotrophic growth).

The bioreactor or fermentor is used to culture microalgal cells throughvarious phases of their physiological cycle. Bioreactors offer manyadvantages for use in the heterotrophic growth and propagation. Toproduce biomass for lipid production, microalgae are preferably grown inlarge quantities in liquid, such as in suspension cultures as anexample. Bioreactors such as stainless steel fermentors can accommodatevery large culture volumes (40,000 liter and greater capacitybioreactors are used in various embodiments of the invention).Bioreactors also typically allow for the control of culture conditionssuch as temperature, pH, oxygen tension, and carbon dioxide levels. Forexample, bioreactors are typically configurable, for example, usingports attached to tubing, to allow gaseous components, like oxygen ornitrogen, to be bubbled through a liquid culture. Other cultureparameters, such as pH of the culture media, the identity andconcentration of trace elements, and other media constituents can alsobe more readily manipulated using a bioreactor.

Bioreactors can be configured to flow culture media through thebioreactor throughout the time period during which the microalgaereproduce and increase in number. In some embodiments, for example,media can be infused into the bioreactor after inoculation but beforethe cells reach a desired density. In other instances, a bioreactor isfilled with culture media at the beginning of a culture, and no moreculture media is infused after the culture is inoculated. In otherwords, the microalgal biomass is cultured in an aqueous medium for aperiod of time during which the microalgae reproduce and increase innumber; however, quantities of aqueous culture medium are not flowedthrough the bioreactor throughout the time period. Thus, in someembodiments, aqueous culture medium is not flowed through the bioreactorafter inoculation.

Bioreactor ports can be used to introduce, or extract gases, solids,semisolids, and liquids, into the bioreactor chamber containingmicroalgae. While many bioreactors have more than one port (for example,one for media entry, and another for sampling), it is not necessary thatonly one substance enter or leave a port. For example, a port can beused to flow culture media or additional carbon source into thebioreactor and later be used for sampling, gas entry, gas exit, or otherpurposes. Preferably, a sampling port can be used repeatedly withoutaltering or compromising the axenic nature of the culture. A samplingport can be configured with a valve or other device that allows the flowof sample to be stopped or started or to provide a means of continoussampling. Bioreactors typically have at least one port that allowsinoculation of a culture, and such a port can also be used for otherpurposes such as media or gas entry.

Bioreactors allow the gas content of the culture of microorganism (e.g.,microalgae) to be manipulated. To illustrate, part of the volume of abioreactor can be gas rather than liquid, and the gas inlets of thebioreactor allow pumping of gases into the bioreactor. Gases that can bebeneficially pumped into a bioreactor include air, air/CO₂ mixtures,noble gases, such as argon, and other gases. Bioreactors are typicallyequipped to enable the user to control the rate of entry of a gas intothe bioreactor. As noted above, increasing gas flow into a bioreactorcan be used to increase mixing of the culture.

Increased gas flow affects the turbidity of the culture as well.Turbulence can be achieved by placing a gas entry port below the levelof the aqueous culture media so that gas entering the bioreactor bubblesto the surface of the culture. One or more gas exit ports allow gas toescape, thereby preventing pressure buildup in the bioreactor.Preferably a gas exit port leads to a “one-way” valve that preventscontaminating microorganisms from entering the bioreactor.

B. Media

Culture media for the cultivation of microorganisms, includingmicroalgae, typically contains components such as a fixed nitrogensource, a fixed carbon source, trace elements, optionally a buffer forpH maintenance, and phosphate (typically provided as a phosphate salt).Other components can include salts such as sodium chloride, particularlyfor seawater microalgae. Nitrogen sources include organic and inorganicnitrogen sources, including, for example, but without limitation,molecular nitrogen, nitrate, nitrate salts, ammonia (pure or in saltform, such as (NH₄)₂SO₄ and NH₄OH), protein (and amino acids), soybeanmeal, cornsteep liquor, and yeast extract. Examples of trace elementsinclude zinc, boron, cobalt, copper, manganese, and molybdenum, in forexample, the respective forms of ZnCl₂, H₃BO₃, CoCl₂. 6H₂O, CuCl₂. 2H₂O,MnCl₂. 4H₂O and (NH₄)₆MO₇O₂₄. 4H₂O.

Solid and liquid growth media are generally available from a widevariety of sources, and instructions for the preparation of particularmedia that is suitable for a wide variety of strains of microorganismscan be found, for example, online at http://www.utex.org/, a sitemaintained by the University of Texas at Austin, 1 University StationA6700, Austin, Tex. 78712-0183, for its culture collection of algae(UTEX). For example, various fresh water and salt water media includethose described in PCT Pub No. 2008/151149, incorporated herein byreference.

In a particular example, Proteose Medium is suitable for axeniccultures, and a 1 liter volume of Proteose Medium (pH ˜6.8) can beprepared by the addition of 1 g proteose peptone to 1 liter of BristolMedium. Bristol Medium comprises 2.94 mM NaNO₃, 0.17 mM CaCl₂.2H₂O, 0.3mM MgSO₄.7H₂O, 0.43 mM K₂HPO₄, 1.29 mM KH₂PO₄ and 1.43 mM NaCl in anaqueous solution. The solution is covered and autoclaved, and the storedat a refrigerated temperature prior to use. Another example is thePrototheca isolateion medium (PIM), which comprises 10 g/L postassiumhydrogen phthalate (KHP), 0.9 g/L sodium hydroxide, 0.1 g/L magnesiumsulfate, 0.2 g/L potassium hydrogen phosphate, 0.3 g/L ammoniumchloride, 10 g/L glucose, 0.001 g/L thiamine hydrochloride, 20 g/L agar,0.25 g/L 5-fluorocytosine, at a pH in the range of 5.0 to 5.2 (see Pore(1973) App. Microbiology, 26:648-649). Other suitable media for use withthe methods of the invention can be readily identified by consulting theURL identified above, or by consulting other organizations that maintaincultures of microorganisms, such as SAG, CCAP or CCALA. SAG refers tothe Culture Collection of Algae at the University of Göttingen(Göttingen, Germany), CCAP refers to the culture collection of algae andprotozoa managed by the Scottish Association for Marine Science(Scotland, United Kingdom), and CCLA refers to the culture collection ofalgal laboratory at the Institute of Botany (T{hacek over (r)}ebo{hacekover (n)}, Czech Republic). Additionally, U.S. Pat. No. 5,900,370describes media formulations and conditions suitable for heterotrophicfermentation of Prototheca species.

For oil (lipid) production, selection of fixed carbon source isimportant, as the cost of the fixed carbon source must be sufficientlylow to make oil production economical. Thus, while suitable carbonsources include, for example, acetate, floidoside, fructose, galactose,glucuronic acid, glucose, glycerol, lactose, mannose,N-acetylglucosamine, rhamnose, sucrose, glucose, and/or xylose,selection of feedstocks containing these compounds is an importantaspect of the methods of the invention. Some microorganism (e.g.,microalgae) species can grow by utilizing a fixed carbon source such asglucose or acetate in the absence of light. Such growth is known asheterotrophic growth. For Chlorella protothecoides, for example,heterotrophic growth results in high production of biomass andaccumulation of high lipid content in cells. Other suitable feedstocksinclude, for example, black liquor, corn starch, depolymerizedcellulosic material, milk whey, molasses, thick cane juice, potato,sorghum, sucrose, sugar beet, sugar cane, rice and wheat. Carbon sourcescan also be provided as a mixture, such as a mixture of sucrose anddepolymerized sugar beet pulp. The one or more carbon source(s) can besupplied at a concentration of at least about 50 μM, at least about 100μM, at least about 500 μM, at least about 5 mM, at least about 50 mM, atleast about 500 mM, of one or more exogenously provided fixed carbonsource(s). Carbon sources of particular interest for purposes of thepresent invention include cellulose (in a depolymerized form), glycerol,sucrose (in the form of cane juice or molasses) and sorghum.

Some microorganisms naturally grow on or can be engineered to grow on afixed carbon source that is a heterogeneous source of compounds such asmunicipal waste, secondarily treated sewage, wastewater, and othersources of fixed carbon and other nutrients such as sulfates,phosphates, and nitrates. The sewage component serves as a nutrientsource in the production of hydrocarbons, and the culture provides aninexpensive source of hydrocarbons.

In some heterotrophic growth methods, microorganisms can be culturedusing cellulosic biomass as a feedstock. Cellulosic biomass (e.g.,stover, such as corn stover) is inexpensive and readily available;however, attempts to use this material as a feedstock for yeast havefailed. In particular, such feedstock have been found to be inhibitoryto yeast growth, and yeast cannot use the 5-carbon sugars produced fromcellulosic materials (e.g., xylose from hemi-cellulose). By contrast,microalgae can grow on processed cellulosic material. Accordingly,heterotrophic growth methods include a method of culturing a microalgaein the presence of a cellulosic material and/or a 5-carbon sugar.Cellulosic materials generally include:

Component Percent Dry Weight Cellulose 40-60% Hemicellulose 20-40%Lignin 10-30%

Suitable cellulosic materials include residues from herbaceous and woodyenergy crops, as well as agricultural crops, i.e., the plant parts,primarily stalks and leaves, not removed from the fields with theprimary food or fiber product. Examples include corn stover (stalks,leaves, husks, and cobs), wheat straw, and rice straw. Five-carbonsugars that are produced from such materials include xylose.

Chlorella protothecoides, for example, has been shown to exhibit higherlevels of productivity when cultured on a combination of glucose andxylose than when cultured on either glucose or xylose alone. Thissynergistic effect provides a significant advantage in that it allowscultivation of Chlorella on combinations of xylose and glucose, such ascellulosic material.

In another embodiment of the methods of the invention, the carbon sourceis sucrose, including a complex feedstock containing sucrose, such asthick cane juice from sugar cane processing or molasses. In oneembodiment, the culture medium further includes at least one sucroseutilization enzyme. In some cases, the culture medium includes a sucroseinvertase. In one embodiment, the sucrose invertase enzyme is asecretable sucrose invertase enzyme encoded by an exogenous sucroseinvertase gene expressed by the population of microorganisms. Thus, insome cases, the microalgae has been genetically engineered to express asucrose utilization enzyme, such as a sucrose transporter, a sucroseinvertase, a hexokinase, a glucokinase, or a fructokinase.

Complex feedstocks containing sucrose include waste molasses from sugarcane processing; the use of this low-value product of sugar caneprocessing can provide significant cost savings in the production oflipids/oil. Another complex feedstock containing sucrose that is usefulin the methods of the invention is sorghum, including sorghum syrup andpure sorghum. Sorghum syrup is produced from the juice of sweet sorghumcane; its sugar profile consists of mainly glucose (dextrose), fructose,and sucrose.

C. Increasing Yield of Lipids

For the production of lipids/oil in accordance with the methods of theinvention, it is preferable to culture cells in the dark, as is thecase, for example, when using extremely large (40,000 liter or greatercapacity) fermentors that do not allow light to strike the culture. Asan example, an inoculum of lipid-producing microalgal cells areintroduced into the medium; there is a lag period (lag phase) before thecells begin to propagate. Following the lag phase, the propagation rateincreases steadily and enters the log, or exponential phase. Theexponential phase is in turn followed by a slowing of propagation due todecreases in nutrients such as nitrogen, increases in toxic substances,and quorum sensing mechanisms. After this slowing, propagation stops,and cells enter a stationary phase or steady growth state, depending onthe particular environment provided to the cells. For obtaininglipid-rich biomass, the culture is typically harvested well after theend of the exponential phase, which may be terminated early by allowingnitrogen or another key nutrient (other than carbon) to become depleted,forcing the cells to convert the carbon sources, which are present inexcess, to lipid. Culture parameters can be manipulated to optimizetotal oil production, the combination of lipid species produced, and/orproduction of a specific oil.

Process conditions can be adjusted to increase the yield of lipidssuitable for use as biodiesel or other target molecules, and/or toreduce production cost. For example, in certain embodiments, a microbe(e.g., a microalgae) is cultured in the presence of a limitingconcentration of one or more nutrients, such as, for example, nitrogen.This condition tends to increase microbial lipid yield over microbiallipid yield in a culture in which nitrogen is provided in excess. Inparticular embodiments, the increase in lipid yield is at least about:10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500%. Themicrobe can be cultured in the presence of a limiting amount of thenutrient for a portion of the total culture period or for the entireperiod. In particular embodiments, the nutrient concentration is cycledbetween a limiting concentration and a non-limiting concentration atleast twice during the total culture period. In addition, as describedabove, certain fixed carbon feedstocks, such as glycerol, can beemployed to increase the percentage of cell weight that is lipid, inrelation to comparable quantities of other fixed carbon feedstocks.

To increase lipid yield, acetic acid can be employed in the feedstockfor a lipid-producing microbe (e.g., a microalgae). Acetic acid feedsdirectly into the point of metabolism that initiates fatty acidsynthesis (i.e., acetyl-CoA); thus providing acetic acid in the culturecan increase fatty acid production. Generally, the microbe is culturedin the presence of a sufficient amount of acetic acid to increasemicrobial lipid yield, and/or microbial fatty acid yield, specifically,over microbial lipid (e.g., fatty acid) yield in the absence of aceticacid.

In another embodiment, lipid yield is increased by culturing alipid-producing microbe (e.g., microalgae) in the presence of one ormore cofactor(s) for a lipid pathway enzyme (e.g., a fatty acidsynthetic enzyme). Generally, the concentration of the cofactor(s) issufficient to increase microbial lipid (e.g., fatty acid) yield overmicrobial lipid yield in the absence of the cofactor(s). In a particularembodiment, the cofactor(s) are provided to the culture by including inthe culture a microbe (e.g., microalgae) containing an exogenous geneencoding the cofactor(s). Alternatively, cofactor(s) may be provided toa culture by including a microbe (e.g., microalgae) containing anexogenous gene that encodes a protein that participates in the synthesisof the cofactor. In certain embodiments, suitable cofactors include anyvitamin required by a lipid pathway enzyme, such as, for example:biotin, pantothenate. Genes encoding cofactors suitable for use in theinvention or that participate in the synthesis of such cofactors arewell known and can be introduced into microbes (e.g., microalgae), usingconstructs and techniques such as those described above.

D. Microalgal Biomass with High Oil Content

Microalgal biomass with a high percentage of oil/lipid accumulation bydry cell weight has been generated using different methods of culture,which are known in the art. Microalgal biomass with a higher percentageof accumulated oil/lipid is useful in accordance with the presentinvention. Li et al. describe Chlorella vulgaris cultures with up to56.6% lipid by dry cell weight (DCW) in stationary cultures grown underautotrophic conditions using high iron concentrations (Li et al.,Bioresource Technology 99(11):4717-22 (2008)). Rodolfi et al., describeNanochloropsis sp. and Chaetoceros calcitrans cultures with 60% lipid byDCW and 39.8% lipid by DCW, respectively, grown in a photobioreactorunder nitrogen starvation conditions (Rodolfi et al., Biotechnology &Bioengineering (2008) [June 18 Epub ahead of print]). Solovchenko etal., describe Parietochloris incise cultures with approximately 30%lipid accumulation (by DCW) when grown phototrophically and under lownitrogen conditions (Solovchenko et al., Journal of Applied Psychology20:245-251 (2008)). Chlorella protothecoides can produce up to 55% lipid(DCW) grown under certain heterotrophic conditions with nitrogenstarvation (Miao and Wu, Bioresource Technology 97:841-846 (2006)).Other Chlorella species including Chlorella emersonii, Chlorellasorokiniana and Chlorella minutissima have been described to haveaccumulated up to 63% oil (DCW) when grown in stirred tank bioreactorsunder low-nitrogen media conditions (Illman et al., Enzyme and MicrobialTechnology 27:631-635 (2000)). Still higher percent lipid accumulationby dry cell weight has been reported, including 70% lipid (DCW)accumulation in Dumaliella tertiolecta cultures grown in increased NaClconditions (Takagi et al., Journal of Bioscience and Bioengineering101(3): 223-226 (2006)) and 75% lipid accumulation in Botryococcusbraunii cultures (Banerjee et al., Critical Reviews in Biotechnology22(3): 245-279 (2002)).

Microalgal biomass generated by the culture methods described herein anduseful in accordance with the present invention comprises at least 10%microalgal oil by dry weight. In some embodiments, the microalgalbiomass comprises at least 15%, at least 25%, at least 35%, at least45%, at least 55%, or at least 60% microalgal oil by dry weight. In someembodiments, the microalgal biomass contains from 10-90% microalgal oil,from 25-75% microalgal oil, form 40-75% microalgal oil, or from 50-70%microalgal oil by dry weight.

E. Culturing Microorganisms Under Induced Conditions

As described herein, in certain embodiments of the present invention,microorganisms are cultured under induced conditions, i.e., by providinga stimulus. Generally, this is carried out as follows: The microorganismis cultured for a first period of time sufficient to increase the celldensity. Then, the stimulus is provided and the microorganisms arecultured for a second period of time. During the second period of timethe desired effect of the stimulus takes place, e.g., induction of anexogenous gene or increased lipid production. Culturing themicroorganisms for the second period of time can be in the continuedpresence of the stimulus. Alternatively, the stimulus may not beprovided or may only partially provided (i.e., for a limited time)during the culturing of the microorganism for the second period of time.

F. Storing Microorganisms Prior to Extraction of Lipid

In some cases it is desirable to store cultured microorganisms for aperiod of time prior to subjecting them to the extraction processesdescribed below. In some methods of the invention, the microorganisms,produced via a culturing process as described herein are optionallystored for a period of time between termination of the culturing processand lysing the microorganism. In some cases, the microorganism is storedfor at least one hour between termination of the culturing process andlysing the cultured microorganism. In other cases, the microorganism isstored for at least two hours, at least three hours, at least fourhours, at least five hours, at least six hours, at least seven hours, atleast eight hours, at least nine hours, at least ten hours, at leasteleven hours, at least twelve hours, at least thirteen hours, at leastfourteen hours, at least fifteen hours, at least sixteen hours, at leastseventeen hours, at least eighteen hours, at least nineteen hours, atleast twenty hours, at least twenty-one hours, at least twenty-twohours, at least twenty-three hours, or for at least twenty-four hoursbetween termination of the culturing process and lysing the culturedmicroorganism. In some cases, the microorganism is stored for at leastthirty-six hours between termination of the culturing process and lysingthe cultured microorganism. In some cases, the microorganism is storedfor at least forty-eight hours between termination of the culturingprocess and lysing the cultured microorganism, or for longer periods oftime. In other cases, the microorganism is stored for at least sixty orseventy-two hours, or longer, between termination of the culturingprocess and lysing the cultured microorgansim.

Microorganisms prepared in a culture process are optionally stored at atemperature below 15 degrees Celsius between termination of theculturing process and lysing the cultured microorganism. In some casesthe microorganisms are stored at a temperature below 14° C., below 13°C., below 12° C., below 11° C., below 10° C., below 9° C., below 8° C.,below 7° C., below 6° C., below 5° C., below 4° C., below 3° C., below2° C., or below 1° C. between termination of the culturing process andlysing the cultured microorganism. In some cases, the microorganism isstored at a temperature above 30 degrees Celsius between termination ofthe culturing process and lysing the cultured microorganism. In somecases, the microorganism is stored at a temperature above 40 degreesCelsius between termination of the culturing process and lysing thecultured microorganism. In other cases, the microorganism is stored at atemperature above 31° C., above 32° C., above 33° C., above 34° C.,above 35° C., above 36° C., above 37° C., above 38° C., above 39° C.,above 41° C., above 42° C., above 43° C., above 44° C., above 45° C.,above 46° C., above 47° C., above 48° C., above 49° C., or above 50° C.between termination of the culturing process and lysing the culturedmicroorganism.

In some cases, storage or ageing of the cultured microorganisms is usedto disrupt the cells to facilitate oil extraction. With storage, thecell structure may weaken sufficiently to cause the contents of thecells to begin leaking, or to permit passage of reagents or othermaterials into the cells to facilitate extraction of the lipid contents.Storage can be used in this context in combination with other lysingmethods described below.

In some cases, the microorganism is subjected to agitation duringstorage. Agitation can be in addition to any combination of storageconditions set forth above. Agitation can be done on a shaker, vortexer,or the like. Agitation can also result from the shear forces presentduring high g force centrifugation. Alternatively, the microorganism isnot agitated during storage. The microorganisms can be stored in abioreactor or other culture vessel, or optionally transferred to aseparate storage container.

VI. Methods of Extraction of Lipid from Microorganism

In one aspect, the present invention is directed to a process forextracting, recovering, isolating or obtaining lipids frommicroorganisms. The process of the present invention is applicable toextracting a variety of lipids from a variety of microorganisms.

A. Lysing Cells

Intracellular lipids produced in microorganisms are extracted afterlysing the cells of the microorganism. Once extracted, the lipids can befurther refined to produce a high purity lipids.

After completion of culturing, the microorganisms can be separated fromthe fermentation broth, preferably without a drying step such as drumdrying, spray drying, tray drying, vacuum drying and other steps thatremove substantially all of the extracellular and intracellular waterfrom the broth. Optionally, the separation is effected by centrifugationto generate a concentrated paste. Centrifugation does not removesignificant amounts of intracellular water from the microorganisms andis not a drying step. The biomass can then be washed with a washingsolution (e.g., DI water) to get rid of the fermentation broth anddebris. Optionally, the washed microbial biomass may also be dried (ovendried, lyophilized, etc.) prior to cell disruption. Alternatively, cellscan be lysed without separation from some or all of the fermentationbroth when the fermentation is complete. For example, the cells can beat a ratio of less than 1:1 v:v cells to extracellular liquid when thecells are lysed.

Microorganism containing a lipid can be lysed to produce a lysate. Asdetailed herein, the step of lysing a microorganism (also referred to ascell lysis) can be achieved by any convenient means, includingheat-induced lysis, adding a base, adding an acid, using enzymes such asproteases and polysaccharide degradation enzymes such as amylases, usingultrasound, mechanical lysis, using osmotic shock, infection with alytic virus, and/or expression of one or more lytic genes. Lysis isperformed to release intracellular molecules which have been produced bythe microorganism. Each of these methods for lysing a microorganism canbe used as a single method or in combination.

The extent of cell disruption can be observed by microscopic analysis.Using one or more of the methods described herein, typically more than70% cell breakage is observed. Preferably, cell breakage is more than80%, more preferably more than 90% and most preferred about 100%.

In particular embodiments, the microorganism is lysed after growth, forexample to increase the exposure of cellular lipid to a catalyst fortransesterification such as a lipase or a chemical catalyst, expressedas described above. The timing of lipase expression (e.g., via aninducible promoter), cell lysis, and the adjustment oftransesterification reaction conditions (e.g., removal of water,addition of alcohol, etc.) can be adjusted to optimize the yield offatty acid esters from lipase-mediated transesterification. Below aredescribed a number of lysis techniques. These techniques can be usedindividually or in combination.

1. Heat-Induced Lysis

In a preferred embodiment of the present invention, the step of lysing amicroorganism comprises heating of a cellular suspension containing themicroorganism. In this embodiment, the fermentation broth containing themicroorganisms (or a suspension of microorganisms isolated from thefermentation broth) is heated until the microorganisms, i.e., the cellwalls and membranes of microorganisms degrade or breakdown. Typically,temperatures applied are at least 50° C. Higher temperatures, such as,at least 60° C., at least 70° C., at least 80° C., at least 90° C., atleast 100° C., at least 110° C., at least 120° C., at least 130° C. orhigher are used for more efficient cell lysis. Example 7 describes anembodiment of lysis using heat treatment.

Lysing cells by heat treatment can be performed by boiling themicroorganism. Alternatively, heat treatment (without boiling) can beperformed in an autoclave (see Example 6). The heat treated lysate maybe cooled for further treatment.

Cell disruption can also be performed by steam treatment, i.e., throughaddition of pressurized steam. Steam treatment of microalgae for celldisruption is described, for example, in U.S. Pat. No. 6,750,048.

2. Lysis Using a Base

In another preferred embodiment of the present invention, the step oflysing a microorganism comprises adding a base to a cellular suspensioncontaining the microorganism.

The base should be strong enough to hydrolyze at least a portion of theproteinaceous compounds of the microorganisms used. Bases which areuseful for solubilizing proteins are known in the art of chemistry.Exemplary bases which are useful in the methods of the present inventioninclude, but are not limited to, hydroxides, carbonates and bicarbonatesof lithium, sodium, potassium, calcium, and mixtures thereof. Apreferred base is KOH. Examples 6 and 7 describe embodiments of celllysis using KOH.

3. Acidic Lysis

In another preferred embodiment of the present invention, the step oflysing a microorganism comprises adding an acid to a cellular suspensioncontaining the microorganism. Acid lysis can be effected using an acidat a concentration of 10-500 mN or preferably 40-160 nM. Acid lysis ispreferably performed at above room temperature (e.g., at 40-160°, andpreferably a temperature of 50-130°. For moderate temperatures (e.g.,room temperature to 100° C. and particularly room temperature to 65°,acid treatment can usefully be combined with sonication or other celldisruption methods. Example 7 describes embodiments of cell lysis usingacidic lysis.

4. Lysing Cells Using Enzymes

In another preferred embodiment of the present invention, the step oflysing a microorganism comprises lysing the microorganism by using anenzyme. Preferred enzymes for lysing a microorganism are proteases andpolysaccharide-degrading enzymes such as hemicellulase (e.g.,hemicellulase from Aspergillus niger; Sigma Aldrich, St. Louis, Mo.;#H2125), pectinase (e.g., pectinase from Rhizopus sp.; Sigma Aldrich,St. Louis, Mo.; #P2401), Mannaway 4.0 L (Novozymes), cellulase (e.g.,cellulose from Trichoderma viride; Sigma Aldrich, St. Louis, Mo.;#C9422), and driselase (e.g., driselase from Basidiomycetes sp.; SigmaAldrich, St. Louis, Mo.; #D9515. Example 7 describes embodiments of celllysis using enzymes.

a) Cellulases

In a preferred embodiment of the present invention, a cellulase forlysing a microorganism is a polysaccharide-degrading enzyme, optionallyfrom Chlorella or a Chlorella virus. Example 7 describes embodiments ofcell lysis using a cellulase. Another example of apolysaccharide-degrading enzyme that can be used is mannaway, asdescribed in the Examples.

b) Proteases

Proteases such as Streptomyces griseus protease, chymotrypsin,proteinase K, proteases listed in Degradation of Polylactide byCommercial Proteases, Oda Y et al., Journal of Polymers and theEnvironment, Volume 8, Number 1, January 2000, pp. 29-32(4), and otherproteases can be used to lyse microorganisms. Other proteases that canbe used include Alcalase 2.4 FG (Novozymes) and Flavourzyme 100 L(Novozymes), as described in the Examples.

c) Combinations

Any combination of a protease and a polysaccharide-degrading enzyme canalso be used, including any combination of the preceding proteases andpolysaccharide-degrading enzymes.

5. Lysis Cells Using Ultrasound

In another preferred embodiment of the present invention, the step oflysing a microorganism is performed by using ultrasound, i.e.,sonication. Thus, cells can also by lysed with high frequency sound. Thesound can be produced electronically and transported through a metallictip to an appropriately concentrated cellular suspension. Thissonication (or ultrasonication) disrupts cellular integrity based on thecreation of cavities in cell suspension. Example 6 describes a methodfor cell lysis using ultrasound. Example 7 describes embodiments of celllysis using sonication.

6. Mechanical Lysis

In another preferred embodiment of the present invention, the step oflysing a microorganism is performed by mechanical lysis. Cells can belysed mechanically and optionally homogenized to facilitate hydrocarbon(e.g., lipid) collection. For example, a pressure disrupter can be usedto pump a cell containing slurry through a restricted orifice valve.High pressure (up to 1500 bar) is applied, followed by an instantexpansion through an exiting nozzle. Cell disruption is accomplished bythree different mechanisms: impingement on the valve, high liquid shearin the orifice, and sudden pressure drop upon discharge, causing anexplosion of the cell. The method releases intracellular molecules.

Alternatively, a ball mill can be used. In a ball mill, cells areagitated in suspension with small abrasive particles, such as beads.Cells break because of shear forces, grinding between beads, andcollisions with beads. The beads disrupt the cells to release cellularcontents. Cells can also be disrupted by shear forces, such as with theuse of blending (such as with a high speed or Waring blender asexamples), the french press, or even centrifugation in case of weak cellwalls, to disrupt cells.

7. Lysing Cells by Osmotic Shock (Cytolysis)

In another preferred embodiment of the present invention, the step oflysing a microorganism is performed by applying an osmotic shock. Thiscan be achieved, for example, by centrifuging fermentation broth andresuspending the cell paste in deionized water.

8. Infection with a Lytic Virus

In a preferred embodiment of the present invention, the step of lysing amicroorganism comprises infection of the microorganism with a lyticvirus. A wide variety of viruses are known to lyse microorganismssuitable for use in the present invention, and the selection and use ofa particular lytic virus for a particular microorganism is within thelevel of skill in the art.

For example, paramecium bursaria chlorella virus (PBCV-1) is theprototype of a group (family Phycodnaviridae, genus Chlorovirus) oflarge, icosahedral, plaque-forming, double-stranded DNA viruses thatreplicate in, and lyse, certain unicellular, eukaryotic chlorella-likegreen algae. Accordingly, any susceptible microalgae, such as C.protothecoides, can be lysed by infecting the culture with a suitablechlorella virus. Methods of infecting species of Chlorella with achlorella virus are known. See for example Adv. Virus Res. 2006;66:293-336; Virology, 1999 Apr. 25; 257(1):15-23; Virology, 2004 Jan. 5;318(1):214-23; Nucleic Acids Symp. Ser. 2000; (44):161-2; J. Virol. 2006March; 80(5):2437-44; and Annu. Rev. Microbiol. 1999; 53:447-94.

9. Autolysis (Expression of a Lytic Gene)

In another preferred embodiment of the present invention, the step oflysing a microorganism comprises autolysis. In this embodiment, amicroorganism according to the invention is genetically engineered toproduce a lytic gene that will lyse the microorganism. This lytic genecan be expressed using an inducible promoter, so that the cells canfirst be grown to a desirable density in a fermentor and then harvested,followed by induction of the promoter to express the lytic gene to lysethe cells. In one embodiment, the lytic gene encodes apolysaccharide-degrading enzyme.

In certain other embodiments, the lytic gene is a gene from a lyticvirus. Thus, for example, a lytic gene from a Chlorella virus can beexpressed in a Chlorella, such as C. protothecoides.

Suitable expression methods are described herein with respect to theexpression of a lipase gene. Expression of lytic genes is preferablydone using an inducible promoter, such as a promoter active inmicroalgae that is induced by a stimulus such as the presence of a smallmolecule, light, heat, and other stimuli. Lytic genes from chlorellaviruses are known. For example, see Virology 260, 308-315 (1999); FEMSMicrobiology Letters 180 (1999) 45-53; Virology 263, 376-387 (1999); andVirology 230, 361-368 (1997).

10. Pressure Oscillation

In another preferred embodiment of the present invention, the step oflysing a microorganism comprises subjecting the microorganism to rapidincreases and decreases in pressure. Such rapid increases and decreasesare preferably performed across a wide enough differential in pressurethat the cells are not able to self-regulate, and burst as a result.

11. Additional Consideration

When lipids are not extracted immediately after isolating themicroorganism, the isolated microorganisms are typically dried. Drying amicroorganism can be done, e.g., on a drum dryer. Dried microorganismscan be packaged in vacuum-sealed containers to prevent degradation oflipids.

B. Treatment of Cell Lysate with Organic Solvent

A lipid produced by a microorganism is also referred herein as a “firstlipid.” A first lipid may comprise one or more lipids produced by themicroorganism.

A lipid used to extract a first lipid from a microorganism is referredto as a “second lipid.” A preferred second lipid is an oil. An organicsolvent of the present invention comprises a second lipid.

Some methods of the present invention comprise the step of treating alysate with an organic solvent. Typically, the organic solvent is addeddirectly to the lysate without prior separation of the lysatecomponents. After addition of the solvent, the lysate separates eitherof its own accord or as a result of centrifugation or the like intodifferent layers. The layers can include in order of decreasing density:a pellet of heavy solids, an aqueous phase, an emulsion phase, and anoil phase. The emulsion phase is an emulsion of lipids and aqueousphase. Depending on the percentage of organic solvent added with respectto the lysate (w/w or v/v), the force of centrifugation if any, volumeof aqueous media and other factors, either or both of the emulsion andoil phases can be present.

Incubation or treatment of the cell lysate or the emulsion phase withthe organic solvent is performed for a time sufficient to allow thelipid produced by the microorganism to become solubilized in the organicsolvent to form a heterogeneous mixture.

1. Oils

In a preferred embodiment of the present invention, an organic solventis an oil selected from the group consisting of oil from soy, rapeseed,canola, palm, palm kernel, coconut, corn, waste vegetable oil, Chinesetallow, olive, sunflower, cotton seed, chicken fat, beef tallow, porcinetallow, microalgae, macroalgae, Cuphea, flax, peanut, choice whitegrease (lard), Camelina sativa mustard seedcashew nut, oats, lupine,kenaf, calendula, hemp, coffee, linseed, hazelnut, euphorbia, pumpkinseed, coriander, camellia, sesame, safflower, rice, tung oil tree,cocoa, copra, pium poppy, castor beans, pecan, jojoba, jatropha,macadamia, Brazil nuts, and avocado. Also included are fossil oils suchas crude oil or a distillate fraction of a fossil oil.

The amount of organic solvent added to the lysate is typically greaterthan 5% (measured by v/v and/or w/w) of the lysate with which thesolvent is being combined. Thus, a preferred v/v or w/w of the organicsolvent is greater than 5%, at least 6%, at least 7%, at least 10%, atleast 20%, at least 25%, at least 30%. at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, and at least 95% ofthe cell lysate.

2. Other Organic Solvents

Other non-limiting examples of organic solvents which can be used topractice the methods of the present invention include hexane, isohexane,methanol, dodecane, fossil-derived crude oil and distillate fractionsthereof, and supercritical carbon dioxide. Methanol can be anadvantageous organic solvent to use in biodiesel manufacturing becauseit can be used in the transesterification process as well as extractionof oil.

C. Solventless Extraction

Lipids can also be extracted from a lysate without substantial or anyuse of organic solvents by cooling the lysate. In such methods, thelysate is preferably produced by acid treatment in combination withabove room temperature. Sonication can also be used, particularly if thetemperature is between room temperature and 65° C. Such a lysate oncentrifugation or settling can be separated into layers, one of which isan aqueous:lipid layer. Other layers can include a solid pellet, anaqueous layer, and a lipid layer. Lipid can be extracted from theemulsion layer by freeze thawing or otherwise cooling the emulsion asdescribed further below. In such methods, it is not necessary to add anyorganic solvent. If any organic solvent is added, the organic solventcan be below 5% v/v or w/w of the lysate.

D. Agitation or No Agitation of the Heterogeneous Mixture

In a preferred embodiment of the present invention, after producing acell lysate and optionally adding the organic solvent to the cell lysateor to the emulsion phase as described above, the heterogeneous mixtureis agitated. Agitation can be done on a shaker, vortexer, or the like.Agitation can also result from the shear forces present during high gforce centrifugation. Alternatively, the heterogeneous mixture is notagitated.

E. Separation of the Heterogeneous Mixture into a Lipid:Organic SolventComposition and an Aqueous Composition and, Optionally an EmulsifiedComposition or Cell Pellet Composition

The cell lysate, optionally treated with an organic solvent, produced bythe above methods is a heterogeneous mixture including lipids, aqueoussolutions, cell debris and the organic solvent (if added). Theheterogeneous mixture can be separated into multiple layers as describedbelow. The multiple layers can include in order of descending density, apellet of cell debris, an aqueous layer, a lipid:aqueous emulsion layerand a lipid layer. The presence and relative proportions of thedifferent layers depends on the lysis technique, the separationtechnique including centrifugal force, concentration of lipid in themicroorganism and whether an organic solvent is used. The desired lipidcan occur both in a separate lipid layer and in a lipid: aqueous layer.The use of an organic solvent favors formation of a separate lipidlayer. The higher the concentration of the organic solvent relative tothe lysis, the more lipid is likely to be found in a separate lipidlayer rather than in an aqueous:lipid emulsion layer. The organicsolvent, if present, associates with the lipid either in the lipid layeror the emulsion or both. The lipid can be obtained both from a separatelipid layer and from an emulsion layer, as described below.

Cell lysates can be separated into different layers as described aboveby techniques, such as, centrifugation and settling, i.e., allowing thelayers to form spontaneously with time.

F. Separation of Lipid

The separation of lipid from other components present in the lysatedepends on which layers are formed as a result of the separation. If aseparate lipid layer is formed, this layer forms on top of other layersand can be suctioned off, pipetted or decanted or the like from the topof the vessel containing the separated layers. This lipid layer can thenbe used in various applications described below. Likewise solid and/oraqueous layers can be drained off and discarded from the bottom of thevessel. If an emulsion layer is formed, the emulsion can be subjected tofurther extraction to separate lipids in the emulsion from aqueousfluid. In such a circumstance, separate aqueous and/or pellet layers, ifpresent, can be drained off before separating the emulsion. A separatelipid layer if present can also be decanted off; however, such is notnecessary, and the separate lipid layer can facilitate separation of theemulsion into its components.

The emulsion can be separated into its components by washing with afurther volume of organic solvent to extract lipids from the emulsion.Alternatively, the emulsion can be washed with an aqueous solution toremove aqueous fluids from the emulsion. Alternatively, the emulsion canbe subject to cooling below room temperature. Preferably, the emulsionis frozen and rethawed. Freezing is preferably to a temperature of −5°C. to −30° C., and is maintained for at least one hour.

After any of the above treatments, the emulsion is re-separated intocomponent layers by the same techniques as previously described. Thecomponent layers can include a cell pellet, aqueous layer, emulsionlayer and lipid layer as previously described. The component layers canbe separated as previously described. If an emulsion layer is present,the emulsion layer can be subjected to a further round of extractionusing the same procedures described above.

After one or more rounds of separation, the lysate eventually yields alipid layer. The lipid layer can be used in various applications asdescribed below. When any of the rounds of extraction is performed withan organic solvent, that organic solvent remains associated with thelipid layer. Typically, it is not necessary to separate the lipid layerfrom the organic solvent. For example, when the organic solvent is anoil or lipid, the lipid present in the microorganisms can be referred toas a first lipid and the lipid or oil used for extraction can bereferred to as a second lipid. For example, the first lipid can betriacylglycerol from a microalgae and the second lipid can be soy oil.After separation of a lipid:organic solvent layer, (in this casemicroalgal lipid:soy oil) the mixture can for example be transesterifiedor hydrotreated to yield biodiesel or renewable diesel, respectively.

One method of separation that can be used on a lysate, including alysate that contains an emulsion, is the application of shear forces.Following cell lysis, oil may be present as an oil-in-water emulsionwith a small (<5 or <10 micron) droplet size. The emulsion may bestabilized by any number of ampipathic emulsifiers (e.g. oleosins,denatured proteins, phospholipids, fatty acids etc.). All else beingequal, the stability of an emulsion is inversely related to the dropletsize of the included phase. There are a number of procedures providedherein that may be applied to cause oil droplets to coalesce andincrease in size, thus rendering the emulsion more amenable to breakage(e.g. gentle agitation in the presence of certain detergents, or acid orbase). Once an oil-in-water emulsion has been treated so as to weakenthe emulsion (by an increase in droplet size, as described above, and/orby some chemical or physical treatment that decreases the efficacy ofthe emulsifying agent(s)), the droplets need to be physically forcedtogether in order that they may coalesce and phase separation may occur.

One method of forcing the included oil droplets together is bycentrifugation that causes physical crowding by forcing the emulsionthrough a thin film and introducing concomitant shear that may berequired to break the emulsion. A device that can be used in the methodsprovided herein to introduce g-force, thin film and shear is a stackeddisk centrifuge in which the light phase is ejected as a thin filmbetween rapidly rotating disks. The film is preferably between 1micrometer and 1000 micrometers. The film is preferably less than orsimilar to the diameter of the included oil droplets.

In one embodiment, microbial oil-bearing cells in fermentation broth arecentrifuged to reduce the water content of the composition. Cells areruptured by homegenization and/or chemical/enzymatic treatment.Optionally, the oil droplets are forced to coalesce by gentle agitationof the lysate. Phase separation of the composition is then achieved bystacked disk centrifugation.

Another method of separation that can be used alone or in combinationwith other methods described herein is the use of one or moresurfactants to destabilize an emulsion. Destabilization results incoalescence of oil globules into larger clusters and eventually into aphase separated composition of a light layer of oil, and a heavieraquesous phase. Preferred surfactants for destabilizing emulsions ofoil-bearing microbial biomass is oleamide DEA (diethanolamine) (StepanChemical Co.) including but not limited to NINOL 201, laurelamide DEAincluding but not limited to NINOL 96L and NINOL 55L and cocoamide DEAincluding but not limited to NINOL 4000. Methods of oil recovery from alipid emulsion using surfactants are provided in the Examples below.

The lipid composition obtained can be analyzed by a number of methods,including HPLC as described in Example 6.

VII. Method of Producing Fuels Suitable for Use in Diesel Vehicles andJet Engines

Increasing interest is directed to the use of hydrocarbon components ofbiological origin in fuels, such as biodiesel, renewable diesel, and jetfuel, since renewable biological starting materials that may replacefossil ones are available, and the use thereof is desirable. There is anurgent need for methods for producing hydrocarbon components frombiological materials. The present invention fulfills this need byproviding methods for production of biodiesel, renewable diesel, and jetfuel using the lipid:organic solvent composition or the lipids describedherein as a biological material to produce biodiesel, renewable diesel,and jet fuel.

After extraction, the present invention provides the advantage that themicrobial oil and the organic solvent (preferably a plant oil) cantogether be subjected to chemical treatment to manufacture a fuel foruse in diesel vehicles and jet engines. The ability to avoid separationof the organic solvent used for extraction from the microbial oilprovides a significant advantage over traditional methods of oilextraction such as hexane extraction in which hexane must be distilledaway from the microbial oil prior to any further processing steps.

Traditional diesel fuels are petroleum distillates rich in paraffinichydrocarbons. They have boiling ranges as broad as 370° to 780° F.,which are suitable for combustion in a compression ignition engine, suchas a diesel engine vehicle. The American Society of Testing andMaterials (ASTM) establishes the grade of diesel according to theboiling range, along with allowable ranges of other fuel properties,such as cetane number, cloud point, flash point, viscosity, anilinepoint, sulfur content, water content, ash content, copper stripcorrosion, and carbon residue. Technically, any hydrocarbon distillatematerial derived from biomass that meets the appropriate ASTMspecification can be defined as diesel, or as biodiesel.

Diesel fuel can be produced from biomass via several types oftechnologies. Feedstocks for diesel fuels derived from biomass include,but are not limited to, soybean, rape seed, canola, palm, and wastecooking oils, along with animal fats. Starting oils can also be of algalorigin. The lipid:organic solvent layer produced by the method of thepresent invention can serve as feedstock to produce biodiesel andrenewable diesel.

A. Biodiesel

Biodiesel is a liquid which varies in color—between golden and darkbrown—depending on the production feedstock. It is practicallyimmiscible with water, has a high boiling point and low vapor pressure.Biodiesel refers to a diesel-equivalent processed fuel for use indiesel-engine vehicles. Biodiesel is biodegradable and non-toxic. Anadditional benefit of biodiesel over conventional diesel fuel is lowerengine wear.

Typically, biodiesel comprises short chain alkyl esters. Variousprocesses convert biomass or a lipid produced and isolated as describedherein to diesel fuels. A preferred method to produce biodiesel is bytransesterification of a lipid as described herein. A preferred shortchain alkyl ester for use as biodiesel is a methyl ester or ethyl ester.

Biodiesel produced by a method described herein can be used alone orblended with conventional diesel fuel at any concentration in mostmodern diesel-engine vehicles. When blended with conventional dieselfuel (petroleum diesel), biodiesel may be present from about 0.1% toabout 99.9%. Much of the world uses a system known as the “B” factor tostate the amount of biodiesel in any fuel mix. For example, fuelcontaining 20% biodiesel is labeled B20. Pure biodiesel is referred toas B 100.

Biodiesel can also be used as a heating fuel in domestic and commercialboilers. Existing oil boilers may contain rubber parts and may requireconversion to run on biodiesel. The conversion process is usuallyrelatively simple, involving the exchange of rubber parts for syntheticparts due to biodiesel being a strong solvent. Due to its strong solventpower, burning biodiesel will increase the efficiency of boilers.

Biodiesel can be used as an additive in formulations of diesel toincrease the lubricity of pure Ulta-Low Sulfur Diesel (ULSD) fuel, whichis advantageous because it has virtually no sulfur content.

Biodiesel is a better solvent than petrodiesel and can be used to breakdown deposits of residues in the fuel lines of vehicles that havepreviously been run on petrodiesel.

1. Production of Biodiesel

Biodiesel can be produced by transesterification of triglyceridescontained in oil-rich biomass and animal fats. The lipid:organic solventlayer or lipids produced by the method of the present invention canserve as feedstock to produce biodiesel. Thus, in another aspect of thepresent invention a method for producing biodiesel is provided. In apreferred embodiment, the method for producing biodiesel comprises thesteps of (a) lysing a lipid-containing microorganism to produce alysate; (b) treating the lysate with an organic solvent for a period oftime sufficient to allow the lipid from the microorganism to becomesolubilized in the organic solvent, whereinby the organicsolvent-treated lysate forms a heterogeneous mixture; (c) separating theheterogeneous mixture into layers comprising a lipid:organic solventlayer and an aqueous layer; (d) removing the lipid:organic solventcomposition from the aqueous composition, emulsion composition, or cellpellet composition; and (e) transesterifying the lipid:organic solventcomposition, whereby biodiesel is produced. The lipid:organic solventcomposition comprises the organic solvent and the microbial lipids.

Methods for growth of a microorganism, lysing a microorganism to producea lysate, treating the lysate in a medium comprising an organic solventto form a heterogeneous mixture and separating the treated lysate into alipid:organic solvent composition and an aqueous or emulsifiedcomposition have been described above and can also be used in the methodof producing biodiesel.

Lipids and lipid:organic solvent composition, where the organic solventis a triacylglyceride selected as described above can be subjected totransesterification to yield long-chain fatty acid esters useful asbiodiesel. Preferred transesterification reactions are outlined belowand include base catalyzed transesterification and transesterificationusing recombinant lipases.

In a base-catalyzed transesterification process, the triacylglyceridesare reacted with an alcohol, such as methanol or ethanol, in thepresence of an alkaline catalyst, typically potassium hydroxide. Thisreaction forms methyl or ethyl esters and glycerin (glycerol) as abyproduct.

a) General Chemical Process

Animal and plant oils are typically made of triglycerides which areesters of free fatty acids with the trihydric alcohol, glycerol. Intransesterification, the glycerol in a triacylglyceride (TAG) isreplaced with a short-chain alcohol such as methanol or ethanol. Atypical reaction scheme is as follows:

In this scheme, the alcohol is deprotonated with a base to make it astronger nucleophile. Commonly, ethanol or methanol is used in vastexcess (up to 50-fold). Normally, this reaction will proceed eitherexceedingly slowly or not at all. Heat, as well as an acid or base canbe used to help the reaction proceed more quickly. The acid or base arenot consumed by the transesterification reaction, thus they are notreactants but catalysts. Almost all biodiesel has been produced usingthe base-catalyzed technique as it requires only low temperatures andpressures and produces over 98% conversion yield (provided the startingoil is low in moisture and free fatty acids).

Any free fatty acids in the base oil are either converted to soap andremoved from the process, or they are esterified (yielding morebiodiesel) using an acidic catalyst.

The most common form of transesterification uses methanol to producemethyl esters as it is the cheapest alcohol available. Ethanol is usedto produce ethyl ester biodiesel. Higher alcohols, such as isopropanoland butanol can also be used.

A byproduct of the transesterification process is the production ofglycerol. Approximately for every ton of biodiesel produced, 100 kg ofglycerol are produced. This glycerol may be used as a chemical buildingblock, and may also be used as a carbon source to fermentmicroorganisms.

b) Using Recombinant Lipases

Transesterification has also been carried out using an enzyme, such as alipase instead of a base. Lipase-catalyzed transesterification can becarried out, for example, at a temperature between the room temperatureand 80° C., and a mole ratio of the TAG to the lower alcohol of greaterthan 1:1, preferably 2:1, more preferably 3:1, and most preferably about3:1.

Lipases suitable for use in transesterification include, but are notlimited to, those listed in Table 11 below. Other examples of lipasesuseful for transesterification are found in, e.g. U.S. Pat. Nos.4,798,793; 4,940,845 5,156,963; 5,342,768; 5,776,741 and WO89/01032.

TABLE 11 Lipases suitable for use in transesterification. Aspergillusniger lipase ABG73614, Candida antarctica lipase B (novozym-435)CAA83122, Candida cylindracea lipase AAR24090, Candida lipolytica lipase(Lipase L; Amano Pharmaceutical Co., Ltd.), Candida rugosa lipase (e.g.,Lipase-OF; Meito Sangyo Co., Ltd.), Mucor miehei lipase (Lipozyme IM20), Pseudomonas fluorescens lipase AAA25882, Rhizopus japonicas lipase(Lilipase A-10FG) Q7M4U7_1, Rhizomucor miehei lipase B34959, Rhizopusoryzae lipase (Lipase F) AAF32408, Serratia marcescens lipase (SMEnzyme) ABI13521, Thermomyces lanuginosa lipase CAB58509, Lipase P(Nagase ChemteX Corporation), and Lipase QLM (Meito Sangyo Co., Ltd.,Nagoya, Japan)

One challenge to using a lipase for the production of fatty acid esterssuitable for biodiesel is that the price of lipase is much higher thanthe price of sodium hydroxide (NaOH) used by the strong base process.This challenge has been addressed by using an immobilized lipase, whichcan be recycled. However, the activity of the immobilized lipase must bemaintained after being recycled for a minimum number of cycles to allowa lipase-based process to compete with the strong base process in termsof the production cost. Immobilized lipases are subject to poisoning bythe lower alcohols typically used in transesterification. U.S. Pat. No.6,398,707 (issued Jun. 4, 2002 to Wu et al.) describes methods forenhancing the activity of immobilized lipases and regeneratingimmobilized lipases having reduced activity.

2. Standards

The common international standard for biodiesel is EN 14214. ASTM D6751is the most common biodiesel standard referenced in the United Statesand Canada. Germany uses DIN EN 14214 and the UK requires compliancewith BS EN 14214.

Basic industrial tests to determine whether the products conform tothese standards typically include gas chromatography, HPLC, and others.Biodiesel meeting the quality standards is very non-toxic, with atoxicity rating (LD₅₀) of greater than 50 mL/kg.

B. Renewable Diesel

Renewable diesel comprises a mixture of alkanes, such as C10:0, C12:0,C14:0,

C16:0 and C18:0 and thus, are distinguishable from biodiesel. Highquality renewable diesel conforms to the ASTM D 975 standard.

The lipid:organic solvent layer or lipids produced by the method of thepresent invention can serve as feedstock to produce renewable diesel.Thus, in another aspect of the present invention, a method for producingrenewable diesel is provided. Renewable diesel can be produced by atleast three processes, hydrothermal processing (hydrotreating),hydroprocessing, and indirect liquefaction. These processes yieldnon-ester distillates. During these processes, triacylglyceridesproduced and isolated as described herein, are broken down to alkenes ofC16 and C18.

Thus, in another aspect of the present invention a method for producingrenewable diesel is provided. In a preferred embodiment, the method forproducing renewable diesel comprises the steps of A method of producingrenewable diesel comprising the steps of: (a) lysing a lipid-containingmicroorganism to produce a lysate; (b) treating the lysate with anorganic solvent for a period of time sufficient to allow the lipid fromthe microorganism to become solubilized in the organic solvent to form aheterogeneous mixture; (c) separating the heterogeneous mixture into alipid:organic solvent composition and an aqueous composition and,optionally, an emulsified composition or cell pellet composition; (d)removing the lipid:organic solvent composition from the aqueouscomposition, emulsion composition, or cell pellet composition; and (e)treating the lipid:organic to produce a straight chain alkane, wherebyrenewable diesel is produced. The lipid:organic solvent compositioncomprises the organic solvent and the cellular lipids.

1. Hydrotreating

In a preferred embodiment of the method for producing renewable diesel,treating the lipid:organic solvent composition or the lipids producedand isolated to produce a straight chain alkane, is performed byhydrotreating of the lipid:organic solvent composition. In hydrothermalprocessing, typically, biomass is reacted in water at an elevatedtemperature and pressure to form oils and residual solids. Conversiontemperatures are typically 570° to 660° F., with pressure sufficient tokeep the water primarily as a liquid, 100 to 170 standard atmosphere(atm). Reaction times are on the order of 15 to 30 minutes. After thereaction is completed, the organics are separated from the water.Thereby a distillate suitable for diesel is produced.

2. Hydroprocessing

A renewable diesel, referred to as “green diesel” can be produced fromfatty acids by traditional hydroprocessing technology. Thetriglyceride-containing oils can be hydroprocessed either as co-feedwith petroleum or as a dedicated feed. The product is a premium dieselfuel containing no sulfur and having a cetane number of 90-100. Thus, inanother preferred embodiment of the method for producing renewablediesel, treating the lipid:organic solvent composition or the lipidsproduced and isolated to produce a straight chain alkane, is performedby hydroprocessing of the lipid:organic solvent composition.

Petroleum refiners use hydroprocessing to remove impurities by treatingfeeds with hydrogen. Hydroprocessing conversion temperatures aretypically 600° to 700° F. Pressures are typically 40 to 100 atm. Thereaction times are on the order of 10 to 60 minutes.

Solid catalysts are employed to increase certain reaction rates, improveselectivity for certain products, and optimize hydrogen consumption.

Hydrotreating and hydroprocessing can ultimately lead to a reduction inthe molecular weight of the feed. In the case of triglyceride-containingoils, the triglyceride molecule is reduced to four hydrocarbon moleculesunder hydroprocessing conditions: a propane molecule and threehydrocarbon molecules, typically in the C12 to C18 range. In somemethods, the first step of treating a triglyceride is hydroprocessing tosaturate double bonds, followed by deoxygenation at elevated temperaturein the presence of hydrogen and a catalyst. In some methods,hydrogenation and deoxygenation occur in the same reaction. In othermethods deoxygenation occurs before hydrogenation. Isomerization is thenoptionally performed, also in the presence of hydrogen and a catalyst.Finally, gases and naphtha components can be removed if desired. Forexample, see U.S. Pat. Nos. 5,475,160 (hydrogenation of triglycerides);5,091,116 (deoxygenation, hydrogenation and gas removal); 6,391,815(hydrogenation); and 5,888,947 (isomerization).

3. Indirect Liquefaction

A traditional ultra-low sulfur diesel can be produced from any form ofbiomass by a two-step process. First, the biomass is converted to asyngas, a gaseous mixture rich in hydrogen and carbon monoxide. Then,the syngas is catalytically converted to liquids. Typically, theproduction of liquids is accomplished using Fischer-Tropsch (FT)synthesis. This technology applies to coal, natural gas, and heavy oils.Thus, in yet another preferred embodiment of the method for producingrenewable diesel, treating the lipid:organic solvent composition or thelipids produced and isolated to produce a straight chain alkane, isperformed by indirect liquefaction of the lipid:organic solventcomposition.

C. Jet Fuel

The annual U.S. usage of jet fuel in 2006 was about 21 billion gallons(about 80 billion liters). Aeroplane fuel is clear to straw colored. Themost common fuel is an unleaded/paraffin oil-based fuel classified asAeroplane A-1, which is produced to an internationally standardized setof specifications. Aeroplane fuel is a mixture of a large number ofdifferent hydrocarbons, possibly as many as a thousand or more. Therange of their sizes (molecular weighs or carbon numbers) is restrictedby the requirements for the product, for example, freezing point orsmoke point. Kerosone-type Aeroplane fuel (including Jet A and Jet A-1)has a carbon number distribution between about 8 and 16 carbon numbers.Wide-cut or naphta-type Aeroplane fuel (including Jet B) has a carbonnumber distribution between about 5 and 15 carbon numbers.

Both Aeroplanes (Jet A and jet B) may contain a number of additives.Useful additives include, but are not limited to, antioxidants,antistatic agents, corrosion inhibitors, and fuel system icing inhibitor(FSII) agents. Antioxidants prevent gumming and usually, are based onalkylated phenols, for example, AO-30, AO-31, or AO-37. Antistaticagents dissipate static electricity and prevent sparking. Stadis 450with dinonylnaphthylsulfonic acid (DINNSA) as the active ingredient, isan example. Corrosion inhibitors, e.g., DCI-4A is used for civilian andmilitary fuels and DCI-6A is used for military fuels. FSII agents,include, e.g., Di-EGME.

A solution is blending algae fuels with existing jet fuel. The presentinvention provides such a solution. The lipid:organic solvent layer orlipids produced by the method of the present invention can serve asfeedstock to produce jet fuel. Thus, in another aspect of the presentinvention, a method for producing jet fuel is provided. Herewith twomethods for producing jet fuel from the lipid:organic solvent layer orlipids produced by the method of the present invention are provided,fluid catalytic cracking (FCC) and hydrodeoxygenation (HDO).

1. Fluid Catalytic Cracking

Fluid Catalytic Cracking (FCC) is one method which is used to produceolefins, especially propylene from heavy crude fractions. There arereports in the literature that vegetable oils such as canola oil couldbe processed using FCC to give a hydrocarbon stream useful as a gasolinefuel.

The lipid:organic solvent layer or lipids produced by the method of thepresent invention can be converted to C₂-C₅ olefins. The processinvolves flowing the lipid:organic solvent layer or lipids producedthrough an FCC zone and collecting a product stream comprised ofolefins, which is useful as a jet fuel. The lipid:organic solvent layeror lipids produced are contacted with a cracking catalyst at crackingconditions to provide a product stream comprising C₂-C₅ olefins andhydrocarbons useful as jet fuel.

Thus, in yet another aspect of the present invention a method forproducing jet fuel is provided. In a preferred embodiment, the methodfor producing jet fuel comprises the steps of (a) lysing alipid-containing microorganism to produce a lysate; (b) treating thelysate with anorganic solvent for a period of time sufficient to allowthe lipid from the microorganism to become solubilized in the organicsolvent to form a heterogeneous mixture;

(c) separating the heterogeneous mixture into a lipid:organic solventcomposition and an aqueous composition and, optionally, an emulsifiedcomposition or cell pellet composition; (d) removing the lipid:organicsolvent composition from the aqueous composition, emulsion composition,or cell pellet composition; and (e) treating the lipid:organic solventcomposition to produce a C2-C5 olefine, whereby jet fuel is produced.The lipid:organic solvent composition comprises the organic solvent andcellular lipids.

In a preferred embodiment of the method for producing a jet fuel, step(f) is performed by flowing the lipid:organic solvent compositionthrough a fluid catalytic cracking zone. Step (f) may further comprisecontacting the lipid:organic solvent composition with a crackingcatalyst at cracking conditions to provide a product stream comprisingC₂-C₅ olefins.

In certain embodiments of this method it may be desirable to remove anycontaminants that may be present in the lipid:organic solventcomposition. Thus, prior to step (f) flowing the lipid:organic solventcomposition through a fluid catalytic cracking zone, the lipid:organicsolvent composition is pretreated. Pretreatment may involve contactingthe lipid:organic solvent composition with an ion-exchange resin. Theion exchange resin is an acidic ion exchange resin, such asAmberlyst™-15 and can be used as a bed in a reactor through which thelipid:organic solvent composition is flowed through, either upflow ordownflow. Other pretreatments may include mild acid washes by contactingthe lipid:organic solvent composition with an acid, such as sulfuric,acetic, nitric, or hydrochloric acid. Contacting is done with a diluteacid solution usually at ambient temperature and atmospheric pressure.

The lipid:organic solvent composition, optionally pretreated, is flowedto an FCC zone where the hydrocarbonaceous components are cracked toolefins. Catalytic cracking is accomplished by contacting thelipid:organic solvent composition in a reaction zone with a catalystcomposed of finely divided particulate material. The reaction iscatalytic cracking, as opposed to hydrocracking, and is carried out inthe absence of added hydrogen or the consumption of hydrogen. As thecracking reaction proceeds, substantial amounts of coke are deposited onthe catalyst. The catalyst is regenerated at high temperatures byburning coke from the catalyst in a regeneration zone. Coke-containingcatalyst, referred to herein as “coked catalyst”, is continuallytransported from the reaction zone to the regeneration zone to beregenerated and replaced by essentially coke-free regenerated catalystfrom the regeneration zone. Fluidization of the catalyst particles byvarious gaseous streams allows the transport of catalyst between thereaction zone and regeneration zone. Methods for cracking hydrocarbons,such as those of the lipid:organic solvent composition described herein,in a fluidized stream of catalyst, transporting catalyst betweenreaction and regeneration zones, and combusting coke in the regeneratorare well known by those skilled in the art of FCC processes. ExemplaryFCC applications and catalysts useful for cracking the lipid:organicsolvent composition to produce C₂-C₅ olefins are described in U.S. Pat.Nos. 6,538,169, 7,288,685, which are incorporated in their entirety byreference.

In one embodiment, cracking the lipid:organic solvent composition of thepresent invention, takes place in the riser section or, alternatively,the lift section, of the FCC zone. The lipid:organic solvent compositionis introduced into the riser by a nozzle resulting in the rapidvaporization of the lipid:organic solvent composition. Before contactingthe catalyst, the lipid:organic solvent composition will ordinarily havea temperature of about 149° C. to about 316° C. (300° F. to 600° F.).The catalyst is flowed from a blending vessel to the riser where itcontacts the lipid:organic solvent composition for a time of abort 2seconds or less.

The blended catalyst and reacted lipid:organic solvent compositionvapors are then discharged from the top of the riser through an outletand separated into a cracked product vapor stream including olefins anda collection of catalyst particles covered with substantial quantitiesof coke and generally referred to as “coked catalyst.” In an effort tominimize the contact time of the lipid:organic solvent composition andthe catalyst which may promote further conversion of desired products toundesirable other products, any arrangement of separators such as aswirl arm arrangement can be used to remove coked catalyst from theproduct stream quickly. The separator, e.g. swirl arm separator, islocated in an upper portion of a chamber with a stripping zone situatedin the lower portion of the chamber. Catalyst separated by the swirl armarrangement drops down into the stripping zone. The cracked productvapor stream comprising cracked hydrocarbons including light olefins andsome catalyst exit the chamber via a conduit which is in communicationwith cyclones. The cyclones remove remaining catalyst particles from theproduct vapor stream to reduce particle concentrations to very lowlevels. The product vapor stream then exits the top of the separatingvessel. Catalyst separated by the cyclones is returned to the separatingvessel and then to the stripping zone. The stripping zone removesadsorbed hydrocarbons from the surface of the catalyst bycounter-current contact with steam.

Low hydrocarbon partial pressure operates to favor the production oflight olefins. Accordingly, the riser pressure is set at about 172 to241 kPa (25 to 35 psia) with a hydrocarbon partial pressure of about 35to 172 kPa (5 to 25 psia), with a preferred hydrocarbon partial pressureof about 69 to 138 kPa (10 to 20 psia). This relatively low partialpressure for hydrocarbon is achieved by using steam as a diluent to theextent that the diluent is 10 to 55 wt-% of lipid:organic solventcomposition and preferably about 15 wt-% of lipid:organic solventcomposition. Other diluents such as dry gas can be used to reachequivalent hydrocarbon partial pressures.

The temperature of the cracked stream at the riser outlet will be about510° C. to 621° C. (950° F. to 1150° F.). However, riser outlettemperatures above 566° C. (1050° F.) make more dry gas and moreolefins. Whereas, riser outlet temperatures below 566° C. (1050° F.)make less ethylene and propylene. Accordingly, it is preferred to runthe FCC process at a preferred temperature of about 566° C. to about630° C., preferred pressure of about 138 kPa to about 240 kPa (20 to 35psia). Another condition for the process is the catalyst tolipid:organic solvent composition ratio which can vary from about 5 toabout 20 and preferably from about 10 to about 15.

In one embodiment of the method for producing a jet fuel, thelipid:organic solvent composition is introduced into the lift section ofan FCC reactor. The temperature in the lift section will be very hot andrange from about 700° C. (1292° F.) to about 760° C. (1400° F.) with acatalyst to lipid:organic solvent composition ratio of about 100 toabout 150. It is anticipated that introducing the lipid:organic solventcomposition into the lift section will produce considerable amounts ofpropylene and ethylene.

Gas and liquid hydrocarbon products produced can be analyzed by gaschromatography, HPLC, etc.

2. Hydrodeoxygenation

In another embodiment of the method for producing a jet fuel using thelipid:organic solvent composition or the lipids produced as describedherein, the structure of the lipid:organic solvent composition or thelipids is broken by a process referred to as hydrodeoxygenation (HDO).As such step (0 is performed by hydrodeoxygenating the lipid:organicsolvent composition.

HDO means removal of oxygen by means of hydrogen, that is, oxygen isremoved while breaking the structure of the material. Olefinic doublebonds are hydrogenated and any sulphur and nitrogen compounds areremoved. Sulphur removal is called hydrodesulphurization (HDS).Pretreatment and purity of the raw materials (lipid:organic solventcomposition or the lipids) contribute to the service life of thecatalyst.

Generally in the HDO/HDS step, hydrogen is mixed with the feed stock(lipid:organic solvent composition or the lipids) and then the mixtureis passed through a catalyst bed as a co-current flow, either as asingle phase or a two phase feed stock. After the HDO/MDS step, theproduct fraction is separated and passed to a separate isomerzationreactor. An isomerization reactor for biological starting material isdescribed in the literature (FI 100 248) as a co-current reactor.

The process for producing a fuel by hydrogenating a hydrocarbon feed,e.g., the lipid:organic solvent composition or the lipids herein, canalso be performed by passing the lipid:organic solvent composition orthe lipids as a co-current flow with hydrogen gas through a firsthydrogenation zone, and thereafter the hydrocarbon effluent is furtherhydrogenated in a second hydrogenation zone by passing hydrogen gas tothe second hydrogenation zone as a counter-current flow relative to thehydrocarbon effluent. Exemplary HDO applications and catalysts usefulfor cracking the lipid:organic solvent composition to produce C₂-C₅olefins are described in U.S. Pat. No. 7,232,935 which is incorporatedin its entirety by reference.

Typically, in the hydrodeoxygenation step, the structure of thebiological component, such as the lipid:organic solvent composition orlipids herein, is decomposed, oxygen, nitrogen, phosphorus and sulphurcompounds, and light hydrocarbons as gas are removed, and the olefinicbonds are hydrogenated. In the second step of the process, i.e. in theso-called isomerization step, isomerzation is carried out for branchingthe hydrocarbon chain and improving the performance of the paraffin atlow temperatures.

In the first step i.e. HDO step of the cracking process, hydrogen gasand the lipid:organic solvent composition or lipids herein which are tobe hydrogenated are passed to a HDO catalyst bed system either asco-current or counter-current flows, said catalyst bed system comprisingone or more catalyst bed(s), preferably 1-3 catalyst beds. The HDO stepis typically operated in a co-current manner. In case of a HDO catalystbed system comprising two or more catalyst beds, one or more of the bedsmay be operated using the counter-current flow principle.

In the HDO step, the pressure varies between 20 and 150 bar, preferablybetween 50 and 100 bar, and the temperature varies between 200 and 500°C., preferably in the range of 300-400° C.

In the HDO step, known hydrogenation catalysts containing metals fromGroup VII and/or VIB of the Periodic System may be used. Preferably, thehydrogenation catalysts are supported Pd, Pt, Ni, NiMo or a CoMocatalysts, the support being alumina and/or silica. Typically,NiMo/Al₂O₃ and CoMo/Al₂O₃ catalysts are used.

Prior to the HDO step, the lipid:organic solvent composition or lipidsherein may optionally be treated by prehydrogenation under milderconditions thus avoiding side reactions of the double bonds. Suchprehydrogenation is carried out in the presence of a prehydrogenationcatalyst at temperatures of 50 400° C. and at hydrogen pressures of 1200 bar, preferably at a temperature between 150 and 250° C. and at ahydrogen pressure between 10 and 100 bar. The catalyst may containmetals from Group VIII and/or VIB of the Periodic System. Preferably,the prehydrogenation catalyst is a supported Pd, Pt, Ni, NiMo or a CoMocatalyst, the support being alumina and/or silica.

A gaseous stream from the HDO step containing hydrogen is cooled andthen carbon monoxide, carbon dioxide, nitrogen, phosphorus and sulphurcompounds, gaseous light hydrocarbons and other impurities are removedtherefrom. After compressing, the purified hydrogen or recycled hydrogenis returned back to the first catalyst bed and/or between the catalystbeds to make up for the withdrawn gas stream. Water is removed from thecondensed liquid. The liquid is passed to the first catalyst bed orbetween the catalyst beds.

After the HDO step, the product is subjected to an isomerization step.It is substantial for the process that the impurities are removed ascompletely as possible before the hydrocarbons are contacted with theisomerization catalyst. The isomerization step comprises an optionalstripping step, wherein the reaction product from the HDO step may bepurified by stripping with water vapour or a suitable gas such as lighthydrocarbon, nitrogen or hydrogen. The optional stripping step iscarried out in counter-current manner in a unit upstream of theisomerization catalyst, wherein the gas and liquid are contacted witheach other, or before the actual isomerization reactor in a separatestripping unit utilizing counter-current principle.

After the stripping step the hydrogen gas and the hydrogenatedlipid:organic solvent composition or lipids herein, and optionally ann-paraffin mixture, are passed to a reactive isomerization unitcomprising one or several catalyst bed(s). The catalyst beds of theisomerization step may operate either in co-current or counter-currentmanner.

It is important for the process that the counter-current flow principleis applied in the isomerization step. In the isomerization step this isdone by carrying out either the optional stripping step or theisomerization reaction step or both in counter-current manner.

The isomerization step and the HDO step may be carried out in the samepressure vessel or in separate pressure vessels. Optionalprehydrogenation may be carried out in a separate pressure vessel or inthe same pressure vessel as the HDO and isomerization steps.

In the isomerzation step, the pressure varies in the range of 20 150bar, preferably in the range of 20 100 bar, the temperature beingbetween 200 and 500° C., preferably between 300 and 400° C.

In the isomerization step, isomerization catalysts known in the art maybe used. Suitable isomerization catalysts contain molecular sieve and/ora metal from Group VII and/or a carrier. Preferably, the isomerizationcatalyst contains SAPO-11 or SAPO41 or ZSM-22 or ZSM-23 or ferrieriteand Pt, Pd or Ni and Al₂O₃ or SiO₂. Typical isomerization catalysts are,for example, Pt/SAPO-11/Al₂O₃, Pt/ZSM-22/Al₂O₃, Pt/ZSM-23/Al₂O₃ andPt/SAPO-11/SiO₂.

As the product, a high quality hydrocarbon component of biologicalorigin, useful as a diesel fuel or a component thereof, is obtained, thedensity, cetane number and performance at low temperate of saidhydrocarbon component being excellent.

VIII. Compositions

Another object of the present invention is to provide compositionscomprising lipids isolated by using the methods described herein. Apreferred composition comprises (i) a first lipid isolated from amicroorganism and (ii) a second lipid, wherein the second lipid isobtained from a source other than the microorganism.

The first lipid can be isolated from any of the microorganisms describedherein.

A. First Lipids

Methods of the present invention are applicable to extracting a varietyof lipids from a variety of microorganisms. Microorganisms describedherein produce a variety of lipids, such as phospholipids, free fattyacids, esters of fatty acids, including triglycerides of fatty acids,sterols; pigments (e.g., carotenoids and oxycarotenoids) and otherlipids, and lipid associated compounds such as phytosterols,ergothionine, lipoic acid and antioxidants including beta-carotene andtocopherol. Exemplary first lipids include, but are not limited to, C8,C10, C12, C14, C16 and C18 triacylglycerides, lipids containing omega-3highly unsaturated fatty acids, such as docosahexaenoic acid (DHA),eicosapentaenoic acid (EPA), and/or docosapentaenoic acid (DPA). Firstlipids also include arachidonic acid, stearidonic acid, cholesterol,desmesterol, astaxanthin, canthaxanthin, and n-6 and n-3 highlyunsaturated fatty acids such as eicosapentaenoic acid, docosapentaenoicacid and docosahexaenoic acid. Other lipids and microorganisms which maybe suitable for use in the instant invention will be readily apparent tothose skilled in the art.

Preferred first lipids are lipids containing a relatively large amountof C18 and C16 fatty acids.

1. C18:1

In a preferred embodiment of the present invention, the first lipid of acomposition comprises at least 50% of a C18:1 lipid, preferably at least60%, and more preferably at least 80%.

2. C10, C12 and C14

In another preferred embodiment of the present invention, the firstlipid of a composition comprises at least 10% of a C10:0, C12:0 andC14:0 lipid combined, preferably at least 15%, more preferably at least20%, and most preferably at least 30%.

B. Second Lipids

The second lipid can be any oil, optionally selected from the groupconsisting of oil from soy, rapeseed, canola, palm, coconut, corn, wastevegetable, Chinese tallow, olive, sunflower, cotton seed. chicken fat,beef tallow, porcine tallow, microalgae, macroalgae, Cuphea, flax,peanut, choice white grease, lard, Camelina sativa, mustard seed, cashewnut, oats, lupine, kenaf, calendula, hemp, coffee, linseed, hazelnuts,euphorbia, pumpkin seed, coriander, camellia, sesame, safflower, rice,tung oil tree, cocoa, copra, pium poppy, castor beans, pecan nuts,jojoba, jatropha, macadamia nuts, Brazil nuts, and avocado. A preferredsecond lipid is coconut oil. Another preferred second lipid is palm oilor soy oil. A second lipid can also be a fossil oil such as crude oil ora distillate fraction of crude oil.

C. Compositions Comprising a First Lipid and a Second Lipid

The first lipid and the second lipid can be provided at differentratios. In a preferred embodiment, the ratio of the first lipid to thesecond lipid is between 1 and 100. In another preferred embodiment, theratio of the first lipid to the second lipid is between 1 and 10. Inanother preferred embodiment, the ratio of the first lipid to the secondlipid is about 1. In another embodiment, the ratio of the second lipidto the first lipid is between 10 and 1.

Additional embodiments of the present invention include optionalfunctional components that would allow one of ordinary skill in the artto perform any of the method variations described herein.

Although the forgoing invention has been described in some detail by wayof illustration and example for clarity and understanding, it will bereadily apparent to one of ordinary skill in the art in light of theteachings of this invention that certain variations, changes,modifications and substitution of equivalents may be made theretowithout necessarily departing from the spirit and scope of thisinvention. As a result, the embodiments described herein are subject tovarious modifications, changes and the like, with the scope of thisinvention being determined solely by reference to the claims appendedhereto. Those of skill in the art will readily recognize a variety ofnon-critical parameters that could be changed, altered or modified toyield essentially similar results.

While each of the elements of the present invention is described hereinas containing multiple embodiments, it should be understood that, unlessindicated otherwise, each of the embodiments of a given element of thepresent invention is capable of being used with each of the embodimentsof the other elements of the present invention and each such use isintended to form a distinct embodiment of the present invention.

The referenced patents, patent applications, and scientific literature,including accession numbers to GenBank database sequences, referred toherein are hereby incorporated by reference in their entirety as if eachindividual publication, patent or patent application were specificallyand individually indicated to be incorporated by reference. Any conflictbetween any reference cited herein and the specific teachings of thisspecification shall be resolved in favor of the latter. Likewise, anyconflict between an art-understood definition of a word or phrase and adefinition of the word or phrase as specifically taught in thisspecification shall be resolved in favor of the latter. The publicationsmentioned herein are cited for the purpose of describing and disclosingreagents, methodologies and concepts that may be used in connection withthe present invention. Nothing herein is to be construed as an admissionthat these references are prior art in relation to the inventionsdescribed herein. In particular, the following patent applications arehereby incorporated by reference in their entireties for all purposes:U.S. Provisional Application No. 61/028,493, filed Feb. 13, 2008,entitled “Extraction of Lipids from Microorganisms”; U.S. ProvisionalApplication No. 61/036,918, filed Mar. 14, 2008, entitled “OilSeparation Methods”; US Provisional Application No. 61/043,318, filedApr. 8, 2008, entitled “Fractionation of Oil-Bearing Biomass”; PCTPatent Application No. PCT/US2009/066142, filed Nov. 30, 2009, entitled“Production of Tailored Oils in Heterotrophic Microorganisms”; and PCTPatent Application No. PCT/US2009/066141, filed Nov. 30, 2009, entitled“Manufacturing of Tailored Oils in Recombinant HeterotrophicMicroorganisms”.

Although this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. The present invention also has a wide variety ofapplications. This application is intended to cover any variations, usesor adaptations of the invention following, in general, the principles ofthe invention and including such departures from the present disclosureas come within known or customary practice within the art to which theinvention pertains and as may be applied to the essential featureshereinbefore set forth.

IX. EXAMPLES Example 1 Cultivation of Microalgae to Achieve High OilContent

Microalgae strains were cultivated to achieve a high percentage of oilby dry cell weight. Cryopreserved cells were thawed at room temperatureand 500 μl of cells were added to 4.5 ml of medium (4.2 g/L K₂HPO₄, 3.1g/L NaH₂PO₄, 0.2 g/L MgSO₄.7H₂O, 0.25 g/L citric acid monohydrate, 0.025g/L CaCl₂.2H₂O, 2 g/L yeast extract) plus 2% glucose and grown for 7days at 28° C. with agitation (200 rpm) in a 6-well plate. Dry cellweights were determined by centrifuging 1 ml of culture at 14,000 rpmfor 5 min in a pre-weighed Eppendorf tube. The culture supernatant wasdiscarded and the resulting cell pellet washed with 1 ml of deionizedwater. The culture was again centrifuged, the supernatant discarded, andthe cell pellets placed at −80° C. until frozen. Samples were thenlyophilized for 24 hours and dry cell weights calculated. Fordetermination of total lipid in cultures, 3 ml of culture was removedand subjected to analysis using an Ankom system (Ankom Inc., Macedon,N.Y.) according to the manufacturer's protocol. Samples were subjectedto solvent extraction with an Ankom XT10 extractor according tomanufacturer's protocol. Total lipid was determined as the difference inmass between acid hydrolyzed dried samples and solvent extracted, driedsamples. Percent oil dry cell weight measurements are shown in Table 12.

TABLE 12 Percent oil dry cell weight for microalgae. Species Strain %Oil Strain # Chlorella kessleri UTEX 387 39.42 4 Chlorella kessleri UTEX2229 54.07 5 Chlorella kessleri UTEX 398 41.67 6 Parachlorella kessleriSAG 11.80 37.78 7 Parachlorella kessleri SAG 14.82 50.70 8 Parachlorellakessleri SAG 21.11 H9 37.92 9 Prototheca stagnora UTEX 327 13.14 10Prototheca moriformis UTEX 1441 18.02 11 Prototheca moriformis UTEX 143527.17 12 Chlorella minutissima UTEX 2341 31.39 13 Chlorellaprotothecoides UTEX 250 34.24 1 Chlorella protothecoides UTEX 25 40.00 2Chlorella protothecoides CCAP 211/8D 47.56 3 Chlorella sp. UTEX 206845.32 14 Chlorella sp. CCAP 211/92 46.51 15 Chlorella sorokiniana SAG211.40B 46.67 16 Parachlorella beijerinkii SAG 2046 30.98 17 Chlorellaluteoviridis SAG 2203 37.88 18 Chlorella vulgaris CCAP 211/11K 35.85 19Chlorella reisiglii CCAP 11/8 31.17 20 Chlorella epllipsoidea CCAP211/42 32.93 21 Chlorella saccharophila CCAP 211/31 34.84 22 Chlorellasaccharophila CCAP 211/32 30.51 23

Example 2 Cultivation of Chlorella protothecoides

Three fermentation processes were performed with three different mediaformulations with the goal of generating algal biomass with high oilcontent. The first formulation (Media 1) was based on medium describedin Wu et al. (1994 Science in China, vol. 37, No. 3, pp. 326-335) andconsisted of per liter: KH₂PO₄, 0.7 g; K₂HPO₄, 0.3 g; MgSO₄-7H₂O, 0.3 g;FeSO₄.7H₂O, 3 mg; thiamine hydrochloride, 10 μg; glucose, 20 g; glycine,0.1 g; H₃BO₃, 2.9 mg; MnCl₂-4H₂O, 1.8 mg; ZnSO₄.7H₂O, 220 μg;CuSO₄.5H₂O, 80 μg; and NaMoO₄.2H₂O, 22.9 mg. The second medium (Media 2)was derived from the flask media described in Example 1 and consisted ofper liter: K₂HPO₄, 4.2 g; NaH₂PO₄, 3.1 g; MgSO₄-7H₂O, 0.24 g; citricacid monohydrate, 0.25 g; calcium chloride dehydrate, 25 mg; glucose, 20g; yeast extract, 2 g. The third medium (Media 3) was a hybrid andconsisted of per liter: K₂HPO₄, 4.2 g; NaH₂PO₄, 3.1 g; MgSO₄.7H₂O, 0.24g; citric acid monohydrate, 0.25 g; calcium chloride dehydrate, 25 mg;glucose, 20 g; yeast extract, 2 g; H₃BO₃, 2.9 mg; MnCl₂-4H₂O, 1.8 mg;ZnSO₄.7H₂O, 220 μg; CuSO₄.5H₂O, 80 μg; and NaMoO₄.2H₂O, 22.9 mg. Allthree media formulations were prepared and autoclave sterilized in labscale fermentor vessels for 30 minutes at 121° C. Sterile glucose wasadded to each vessel following cool down post autoclave sterilization.

Inoculum for each fermentor was Chlorella protothecoides (UTEX 250),prepared in two flask stages using the medium and temperature conditionsof the fermentor inoculated. Each fermentor was inoculated with 10%(v/v) mid-log culture. The three lab scale fermentors were held at 28°C. for the duration of the experiment. The microalgal cell growth inMedia 1 was also evaluated at a temperature of 23° C. For all fermentorevaluations, pH was maintained at 6.6-6.8, agitations at 500 rpm, andairflow at 1 vvm. Fermentation cultures were cultivated for 11 days.Biomass accumulation was measured by optical density at 750 nm and drycell weight.

Lipid/oil concentration was determined using direct transesterificationwith standard gas chromatography methods. Briefly, samples offermentation broth with biomass was blotted onto blotting paper andtransferred to centrifuge tubes and dried in a vacuum oven at 65-70° C.for 1 hour. When the samples were dried, 2 mL of 5% H₂SO₄ in methanolwas added to the tubes. The tubes were then heated on a heat block at65-70° C. for 3.5 hours, while being vortexed and sonicatedintermittently. 2 ml of heptane was then added and the tubes were shakenvigorously. 2Ml of 6% K₂CO₃ was added and the tubes were shakenvigorously to mix and then centrifuged at 800 rpm for 2 minutes. Thesupernatant was then transferred to GC vials containing Na₂SO₄ dryingagent and ran using standard gas chromatography methods. Percentoil/lipid was based on a dry cell weight basis. The dry cell weights forcells grown using: Media 1 at 23° C. was 9.4 g/L; Media 1 at 28° C. was1.0 g/L, Media 2 at 28° C. was 21.2 g/L; and Media 3 at 28° C. was 21.5g/L. The lipid/oil concentration for cells grown using: Media 1 at 23°C. was 3 g/L; Media 1 at 28° C. was 0.4 g/L; Media 2 at 28° C. was 18g/L; and Media 3 at 28° C. was 19 g/L. The percent oil based on dry cellweight for cells grown using: Media 1 at 23° C. was 32%; Media 1 at 28°C. was 40%; Media 2 at 28° C. was 85%; and Media 3 at 28° C. was 88%.The lipid profiles (in area %, after normalizing to the internalstandard) for algal biomass generated using the three different mediaformulations at 28° C. are summarized below in Table 13.

TABLE 13 Lipid profiles for Chlorella protothecoides grown underdifferent media conditions. Media 1 28° C. Media 2 28° C. Media 3 28° C.(in Area %) (in Area %) (in Area %) C14:0 1.40 0.85 0.72 C16:0 8.71 7.757.43 C16:1 — 0.18 0.17 C17:0 — 0.16 0.15 C17:1 — 0.15 0.15 C18:0 3.773.66 4.25 C18:1 73.39  72.72 73.83  C18:2 11.23  12.82 11.41  C18:3alpha 1.50 0.90 1.02 C20:0 — 0.33 0.37 C20:1 — 0.10 0.39 C20:1 — 0.25 —C22:0 — 0.13 0.11

Example 3 Genotyping of Microalgae with High Oil Content

Microalgae samples from the 23 strains listed in Table 12 above weregenotyped. Genomic DNA was isolated from algal biomass as follows. Cells(approximately 200 mg) were centrifuged from liquid cultures for 5minutes at 14,000×g. Cells were then resuspended in sterile distilledwater, centrigured for 5 minutes at 14,000×g and the supernatantdiscarded. A single glass bead ˜2 mm in diameter was added to thebiomass and tubes were placed at −80° C. for at least 15 minutes.Samples were removed and 150 μl of grinding buffer (1% Sarkosyl, 0.25 Msucrose, 50 mM NaCl, 20 mM EDTA, 100 mM Tris-HCl, pH 8.0, RNase A 0.5μg/μl) was added. Pellets were resuspended by vortexing briefly,followed by the addition of 40 μl of 5M NaCl. Samples were vortexedbriefly, followed by the addition of 66 μl of 5% CTAB (Cetyltrimethylammonium bromide) and a final brief vortex. Samples were nextincubated at 65° C. for 10 minutes after which they were centrifuged at14,000×g for 10 minutes. The supernatant was transferred to a fresh tubeand extracted once with 300 μl Phenol: Chloroform:Isoamyl alcohol12:12:1, followed by centrifugation for 5 minutes at 14,000×g. Theresulting aqueous phase was transferred to a fresh tube containing 0.7vol of isoproanol (˜190 μl), mixed by inversion and incubated at roomtemperature for 30 minutes or overnight at 4° C. DNA was recovered viacentrifugation at 14,000×g for 10 minutes. The resulting pellet was thenwashed twice with 70% ethanol, followed by a final wash with 100%ethanol. Pellets were air dried for 20-30 minutes at room temperaturefollowed by resuspension in 50 μl of 10 mM TrisCl, 1 mM EDTA (pH 8.0).

Five μl of total algal DNA, prepared as described above, was diluted1:50 in 10 mM Tris, pH 8.0. PCR reactions, final volume 20 μl, were setup as follows. Ten μl of 2× iProof HF master mix (BIO-RAD) was added to0.4 μl primer SZ02613 (5′-TGTTGAAGAATGAGCCGGCGAC-3′ (SEQ ID NO:5) at 10mM stock concentration). This primer sequence runs from position 567-588in Gen Bank accession no. L43357 and is highly conserved in higherplants and algal plastid genomes. This was followed by the addition of0.4 μl primer SZ02615 (5′-CAGTGAGCTATTACGCACTC-3′ (SEQ ID NO:6) at 10 mMstock concentration). This primer sequence is complementary to position1112-1093 in Gen Bank accession no. L43357 and is highly conserved inhigher plants and algal plastid genomes. Next, 5 μl of diluted total DNAand 3.2 μl dH₂O were added. PCR reactions were run as follows: 98° C.,45″; 98° C., 8″; 53° C., 12″; 72° C., 20″ for 35 cycles followed by 72°C. for 1 mM and holding at 25° C. For purification of PCR products, 20μl of 10 mM Tris, pH 8.0, was added to each reaction, followed byextraction with 40 μl of Phenol:Chloroform:isoamyl alcohol 12:12:1,vortexing and centrifuging at 14,000×g for 5 minutes. PCR reactions wereapplied to S-400 columns (GE Healthcare) and centrifuged for 2 minutesat 3,000×g. Purified PCR products were subsequently TOPO cloned intoPCR8/GW/TOPO and positive clones selected for on LB/Spec plates.Purified plasmid DNA was sequenced in both directions using M13 forwardand reverse primers. Sequences from strains 1-23 (designated in Example1, Table 12) are listed as SEQ ID NOs: 7-29 in the attached SequenceListing.

Example 4 Genomic DNA Analysis of 23S rRNA from Chlorella protothecoidesand Prototheca

Genomic DNA from 8 strains of Chlorella protothecoides (UTEX 25, UTEX249, UTEX 250, UTEX 256, UTEX 264, UTEX 411, CCAP 211/17 and CCAP211/8d) was isolated and genomic DNA analysis of 23S rRNA was performedaccording to the methods described above in Example 3. All strains ofChlorella protothecoides tested were identical in sequence except forUTEX 25. Sequences for all eight strains are listed as SEQ ID NOs: 30and 31 in the attached Sequence Listing.

The 23s rRNA genomic sequence for Prototheca moriformis UTEX 1436 (SEQID NO: 32) was also compared to other Prototheca species (UTEX 1435,UTEX 1437, and UTEX 1439) and the above described Chlorellaprotothecoides strains. The comparison showed that the 23s rRNA genomicsequence for Prototheca moriformis UTEX 1436 was dissimilar to the otherPrototheca genotypes (SEQ ID NO: 33).

Example 5 Diversity of Lipid Chains in Microalgal Species

Lipid samples from a subset of strains grown in Example 1, Table 12 wereanalyzed for lipid profile using HPLC. Results are shown below in Table14.

TABLE 14 Diversity of lipid chains in microalgal species. Strain # fromTable 12 C:14:0 C:16:0 C:16:1 C:18:0 C:18:1 C:18:2 C:18:3 C:20:0 C:20:11 0.57 10.30 0 3.77 70.52 14.24 1.45 0.27 0 2 0.61 8.70 0.30 2.42 71.9814.21 1.15 0.20 0.24 4 0.68 9.82 0 2.83 65.78 12.94 1.46 0 0 5 1.4721.96 0 4.35 22.64 9.58 5.2 3.88 3.3 10 0 12.01 0 0 50.33 17.14 0 0 0 111.41 29.44 0.70 3.05 57.72 12.37 0.97 0.33 0 12 1.09 25.77 0 2.75 54.0111.90 2.44 0 0

Example 6 Extraction of Lipids from Microalgae Using Coconut Oil

1. Cell Production

An F-Tank batch of Chlorella protothecoides (about 1,200 gallons) wasused to generate biomass for extraction processes. The batch (#ZA07126)was allowed to run for 100 hours, while controlling the glucose levelsat 16 g/L, after which time the corn syrup feed was terminated. Residualglucose levels dropped to <0 g/L two hours later. This resulted in afinal age of 102 hours. The final broth volume was 1,120 gallons. Bothin-process contamination checks and a thorough analysis of a final brothsample failed to show any signs of contamination.

2. Cell Disruption

Lyophilized Chlorella protothecoides cells were resuspended to 200 g/Lwith DI water containing 20 g/L KOH. This cell suspension was autoclavedat 130° C. for 30 minutes and cooled to room temperature. A 10 ml sampleof this material was sonicated using a Misonix 3000 sonicator equippedwith a micro-tip on level 7. The suspension was sonicated for 6 minuteson a 30 sec on/off cycle for a total of 3 cycles. More than 70% breakagewas observed by microscope analysis. An aliquot of this suspension (1 mlsample) was centrifuged at 14 K rpm for 15 min. Three layers wereobserved: a pellet of heavy solids, an aqueous phase and an emulsionphase. No oil layer was observed.

A second set of experiments were performed looking at cell breakageefficiency at a variety of times of sonication to achieve maximal cellbreakage. 6 ml samples of Chlorella protothecoides cells prepared asdescribed above were sonicated using a Misonix 3000 sonicator equippedwith a micro-tip on level 8 with interval times of 20 seconds on and 20seconds off. The sonication times ranged from 10 minutes, 20 minutes and30 minutes and were all performed with the samples on ice. The sampleswere then centrifuged at 4300 rpm for 30 minutes. Three layers wereobserved: a pellet of heavy solids, an aqueous phase and an emulsionphase. No oil layer was observed. Each time point sample was alsoanalyzed qualitatively for total cell breakage by looking for wholecells in the lysate under 100× magnification. No whole cells wereobserved in any of the time point samples, indicating that 10 minutes ofsonication was sufficient to achieve maximal cell breakage. Micrographsof the lysate under magnification showed oil droplets and cell debrisconsisting of organelles and disrupted cell membranes. To furtherinspection of the cell pellets from each of the samples showed no wholecells were observed, only cell debris.

3. Algal Oil Purification for Control

Chlorella protothecoides oil was obtained from dried biomass via hexaneextraction using standard methods known in the art (see for example U.S.Pat. Nos. 5,567,732; 6,255,505; and Yamada et al., Industrialapplications of single cell oils, Eds. Kyle and Ratledge, 118-138(1992)).

4. HPLC Analysis of Fatty Acid Profiles

The fatty acid concentrations and profiles of (i) Chlorella cells, (ii)pure algal oil, (iii) pure coconut oil, and (iv) the extracted oilsamples were determined by HPLC.

A 50 μl sample of the (200 g/L) washed Chlorella cell suspension (beforecaustic and heat treatment; see above) was hydrolyzed by incubation at80° C. in an isopropanol/KOH saturated solution for 4 hours. The cellswere then centrifuged at 14 K rpm for 10 min and the hydrolysate wasloaded onto an HPLC column. Samples were analyzed using an Aligent 1100HPLC using the following method. The samples were derivatized withbromophenacyl bromide (60 mg/ml) and loaded onto a Luna 5u C8(2) 100A150×2 mm column (Phenomenex). The samples were eluted from the columnusing a gradient of water to 100% Acetonitrile:tetrahydrofuran (95:5).Signals were detected using DAD array detector at a wavelength of 254nm. The result of such an analysis detailing the lipid profiles for pureoils (algal and coconut oil), established separately (i.e., not mixedwith each other), is shown in FIG. 1. This result demonstrated that thelipid profiles of pure algal oil and coconut oil are different. As such,coconut oil can be used as an organic solvent to extract lipids frommicroalgae and the percent of lipids extracted can be determined (seebelow).

5. Construction and Validation of Theoretical Curves

The fatty acids from mixtures of pure algal oil extracted from Chlorellaand coconut oil were measured as percentages of the total lipid and areshown in Table 15.

TABLE 15 Fatty acid content as percentage of total lipid. % Coconut %Algal Oil Oil C18:1 C12 C14 C18:1/C12 C18:1/C14 0 100 59 0.01 1.00 5,90059.00 2 98 57.9 1.09 8.14 53.13 7.12 5 95 56.3 2.71 8.39 20.76 6.71 1090 53.5 5.41 8.81 9.89 6.08 20 80 48.0 10.81 9.64 4.44 4.98 25 75 45.313.51 10.06 3.35 4.5 40 60 37.0 21.61 11.31 1.71 3.27 50 50 31.5 27.0112.15 1.17 2.59 60 40 26.0 32.40 12.98 0.80 2.00 27 25 17.8 40.5 14.230.44 1.25 80 20 15.0 43.2 14.65 0.35 1.02 89.75 10.25 9.6 48.47 15.460.20 0.62 94.75 5.25 6.9 51.17 15.88 0.13 0.43 100 0 4 54 17.00 0.070.24

In pure algal oil, 59% of the total fatty acids are of the C18:1 type,0.01% are of the C12 type, and 1% are of the C14 type. The remaining39.99% of algal fatty acids are C10:1, C10, C18:3, C18:2, C16 or C18fatty acids, the majority being C18:2 fatty acids (see FIG. 1). In purecoconut oil, 4% of the fatty acids are of the C18:1 type, 54% are of theC12 type, and 17% are of the C14 type. The remaining 25% are C10:1, C10,C18:3, C18:2, C16 or C18 fatty acids (see FIG. 1). Upon mixing algal oilwith coconut oil, the ratio of the respective fatty acids changes. Forexample, in a 1:1 mixture of pure algal oil and pure coconut oil, 31.5%of the total fatty acids are of the C18:1 type (where the majority isfrom algal oil), 27.01% are of the C12 type (which are almostexclusively from coconut oil), and 12.15% are of the C14 type (where themajority is from coconut oil). This reference table is useful fordetermining the percent of algal fatty acids and the percent of coconutfatty acids upon HPLC analysis of a lipid:organic solvent compositionobtained from the lightest layer of a microalgal biomass lysate that hasbeen subjected to centrifugation.

A curve was constructed based on the expected total C18:1 vs C12 or C14in a mixture of the two pure oils, i.e., algal oil and coconut oil. Thetheoretical measurements for both curves were validated by HPLC analysisof a 1:1 mixture of pure algal to pure coconut oil which should resultin 50% of each ratio. The measured value from the C18:1 to C12 and C14was 51% and 65% respectively when fitted to the corresponding curve(FIG. 2).

6. Separating Microbial Oil from an Emulsion Using an Organic Solvent(Coconut Oil Example)

In order to free the oil from the Chlorella lysate, coconut oil wasadded to 1 ml of disrupted Chlorella cell material at ratios of 3:1,1:1, 1:3 and 1:10 (algal oil to coconut oil), mixed well and heated for15 min at 70° C. and then centrifuged for 15 min at 14 K rpm in amicrocentrifuge. Upon centrifugation 4 layers were observed: a pellet ofheavy solids, an aqueous phase, an emulsion phase an oil phase. Then theoil phase was separated from the other layers by pipetting. The oilphase included a mixture of algal oil and coconut oil of which analiquot as subjected to HPLC analysis. The measurement of algal oil inthe suspension was based on oil content of cells. Controls included (i)3 mg of pure algal oil, (ii) 3 mg of pure coconut oil; and (iii) a 1:1mixture of pure algal oil and pure coconut oil. A 5 μl sample from eachwas analyzed by HPLC.

7. Results

The oil content of the starting cell mass (CM) was approximately 30% asdetermined by HPLC. Coconut oil was used to extract lipids from adisrupted cell suspension of Chlorella (200 g/L was suspension) inmixtures of 3:1, 1:1, 1:3 and 1:10 (algal to coconut oil). The samplesin this experiment included: (1) 3 mg pure algal oil (control); (2) 3 mgpure coconut oil (control); (3) 1:1 mix of 3 mg of each pure oil(control); (4) CM (1 ml)+coconut oil (3:1); (5) CM (1 ml)+coconut oil(1:1); (6) CM (1 ml)+coconut oil (1:3); (7) CM (1 ml)+coconut oil(1:10); and (8) CM (1 ml).

No oil layer was present in the sample without coconut oil (8). Thefatty acid composition (expressed as % fatty acid of total lipid) of thepure oil controls and each of the extraction mixtures (experiments(1)-(7)) is shown in Table 16.

TABLE 16 Fatty acid composition of pure oil controls and extractionmixtures. 1:1 Mix of Algal Oil and C.p./ C.p./ % Fatty Coconut CoconutC.oil C.p./ C.oil C.p./ Acid of Algal Oil Oil (3:1) C.oil (1:3) C.oilTotal Oil (1) (2) (3) (4) (1:1) (5) (6) (1:10) (7) C10:1 0% 1% 0% 0% 0%0% 0% C10 0% 10% 5% 9% 10% 9% 10% C12 0% 56% 27% 50% 53% 53% 54% C18:34% 0% 1% 0% 0% 0% 0% C14 1% 18% 9% 18% 17% 18% 18% C18:2 25% 1% 11% 1%1% 1% 1% C16 11% 8% 9% 8% 8% 8% 8% C18:1 59% 4% 33% 11% 8% 8% 6% C18:00% 2% 4% 3% 3% 3% 2%C.p., Chlorella protothecoides; C.oil, coconut oil

The ratios of C18:1 to C12 and to C14 were calculated and fitted intothe theoretical graph (see above and FIG. 2). The extraction efficiencyof the coconut oil was determined by dividing the calculated percent ofalgal oil (from theoretical curve) by the expected percent (oil contentin starting material). The extraction efficiency ranged from 44% in the1:10 (algal oil to coconut oil) mixture (see experiment (7) above) to16% in the 3:1 mixture (see experiment (4) above) as determined from theC18:1 to C12 data (Table 17). Thus, the higher the percent of coconutoil in the extraction procedure, the higher the yield of extractinglipids from Chorella.

TABLE 17 Extraction efficiency as a function of coconut oil fraction. %Algal Oil in Measured % Extraction Extraction C18:1 to C12 % Measured(C18:1/C12)  9 (7) 0.12 4.0 44% 25 (6) 0.14 6.0 24% 50 (5) 0.16 7.0 14%75 (4) 0.22 12.0 16%

The range of the extraction efficiency was 33% (see experiment (7)above) to 14% (see experiment (4) above) as determined from the C18:1 toC14 data (Table 18). Thus, the higher the percent of coconut oil in theextraction procedure, the higher the yield of extracting lipids fromChorella.

TABLE 18 Extraction efficiency as a function of coconut oil fraction. %Algal Oil in Measured % Extraction Extraction C18:1 to C14 % Measured(C18:1/C14)  9 (7) 0.35 3.0 33% 25 (6) 0.43 5.3 21% 50 (5) 0.47 6.3 13%75 (4) 0.62 10.3 14%

The above experiments demonstrated that an organic solvent (in this casecoconut oil) efficiently extracted lipids from an algal cell mass toyield a lipid:organic solvent composition.

An additional set of experiments were performed to look at theefficiency of extracting lipids from a disrupted cell suspension ofChlorella protothecoides (sonication) using four emulsion breakingsurfactants. The surfactants that were used in this study were: oleamideDEA (NINOL 201), laurelamide DEA (NINOL 55LL), laurelamide DEA (NINOL96L) and cocoamide DEA (NINOL 40CO). Each surfactant was added in theconcentrations of 0.2%, 0.5% and 1.0% to the cell lysate suspensions andwere placed in a oven at 55° C. for 15 hours. Each tube was thencentrifuged at 4200 rpm for 20 minutes. In each sample, four phasescould be seen: a solid phase, an aqueous phase, and emulsion layer andan oil layer on the top. Negative control samples with no addedsurfactant produced no visible oil phase, but produced only the solid,aqueous and emulsion layers. The oil layers from each of the surfactanttreated samples were carefully pipetted off and were weighed todetermine overall oil yields. The results are summarized below in Table19. These results show that an organic solvent, such as a surfactant,efficiently extracted lipids from lipid-containing algae biomass toyield a lipid:organic solvent composition.

TABLE 19 Use of surfactants to extract lipids from algae biomass.Surfactant NINOL NINOL NINOL NINOL NINOL NINOL NINOL Name 201 201 20155LL 55LL 55LL 40C0 Surfactant Oleamide Oleamide Oleamide LaurelamidLaurelamid Laurelamid Cocoamide DEA DEA DEA DEA DEA DEA DEA Percent v/v 1.0%  0.5%  0.2%  1.0%  0.5%  0.2%  1.0% Oil in 62 62 62 62 62 62 62Ferm g/L (g) Oil 0.31 0.31 0.31 0.31 0.31 0.31 0.31 in 5 mL sample(theoretical) Temperature 55 55 55 55 55 55 55 (C.) Time (hrs) 15 15 1515 15 15 15 TOTAL Oil in 0.183 0.237 0.041 0.262 0.012 0.026 0.276sample (g) % of 59.07% 76.55% 13.11% 84.44% 4.03% 8.40% 89.01%theoretical Surfactant NINOL NINOL NINOL NINOL NINOL Name 40C0 40C0 96SL96SL 96SL Surfactant Cocoamide Cocoamide Laurelamid LaurelamidLaurelamid DEA DEA DEA DEA DEA Percent v/v  0.5%  0.2%  1.0%  0.5%  0.2%Oil in 62 62 62 62 62 Ferm g/L (g) Oil 0.31 0.31 0.31 0.31 0.31 in 5 mLsample (theoretical) Temperature 55 55 55 55 55 (C.) Time (hrs) 15 15 1515 15 TOTAL Oil in 0.082 0.000 0.286 0.035 0.000 sample (g) % of 26.45%0.00% 92.30% 11.41% 0.00% theoretical

Example 7 Lysing of Chlorella Protothecoides

1. Biomass Production

An F-Tank batch of Chlorella protothecoides (about 1,200 gallons) wasused to generate biomass for extraction processes. The batch (#ZA07126)was allowed to run for 100 hours, while controlling the glucose levelsat 16 g/L, after which time the corn syrup feed was terminated. Residualglucose levels dropped to <0 g/L two hours later. This resulted in afinal age of 102 hours. The final broth volume was 1,120 gallons. Bothin-process contamination checks and a thorough analysis of a final brothsample failed to show any signs of contamination.

2. Heat and Chemical Treatment

Cells were resuspended in water to a biomass concentration of 150 g/Land aliquoted into 27×5 mL tubes. Each tube was conditioned per thematrix below in Table 20.

TABLE 20 Chemical/heat treatment matrix. Tube Condition [KOH] mN [H₂SO₄]mN Temp, 30 min 1 Control 0 0 25° C. 2 40 mN KOH 40 0 25° C. 3 80 mN KOH80 0 25° C. 4 120 mN KOH 120 0 25° C. 5 160 mN KOH 160 0 25° C. 6 40 mNH₂SO₄ 0 40 25° C. 7 80 mN H₂SO₄ 0 80 25° C. 8 120 mN H₂SO₄ 0 120 25° C.9 160 mN H₂SO₄ 0 160 25° C. 10 Control 0 0 65° C. 11 40 mN KOH 40 0 65°C. 12 80 mN KOH 80 0 65° C. 13 120 mN KOH 120 0 65° C. 14 160 mN KOH 1600 65° C. 15 40 mN H₂SO₄ 0 40 65° C. 16 80 mN H₂SO₄ 0 80 65° C. 17 120 mNH₂SO₄ 0 120 65° C. 18 160 mN H₂SO₄ 0 160 65° C. 19 Control 0 0 130° C.20 40 mN KOH 40 0 130° C. 21 80 mN KOH 80 0 130° C. 22 120 mN KOH 120 0130° C. 23 160 mN KOH 160 0 130° C. 24 40 mN H₂SO₄ 0 40 130° C. 25 80 mNH₂SO₄ 0 80 130° C. 26 120 mN H₂SO₄ 0 120 130° C. 27 160 mN H₂SO₄ 0 160130° C.

After treatment, a 1.5 ml sample was centrifuged at 14 K rpm for 15 min,and the size of each of the resulting layers was measured.

3. Emulsion Water Washing

A fresh set of samples were generated under conditions for samples 21-27using a total of 10 mL cell suspension, and aliquoted into 3×3 mLsamples. The resulting emulsions were isolated in new 15 mL tubes, andsubjected to the following water wash scheme according to Table 21.

TABLE 21 Emulsion water washing matrix. Wash 1 Wash 2 Ratio of waterwash Ratio of water wash Sample volume:emulsion volume:emulsion 21 1 to1 1 to 1 22 1 to 1 1 to 1 23 1 to 1 1 to 1 24 1 to 1 1 to 1 25 1 to 1 1to 1 26 1 to 1 1 to 1 27 1 to 1 1 to 1 21 5 to 1 5 to 1 22 5 to 1 5 to 123 5 to 1 5 to 1 24 5 to 1 5 to 1 25 5 to 1 5 to 1 26 5 to 1 5 to 1 27 5to 1 5 to 1 21 10 to 1 10 to 1 22 10 to 1 10 to 1 23 10 to 1 10 to 1 2410 to 1 10 to 1 25 10 to 1 10 to 1 26 10 to 1 10 to 1 27 10 to 1 10 to 1

The water was added to the emulsion, vortexed, and centrifuged at 4,400rpm for 10 minutes. Following the 1st wash, the aqueous phase wasremoved, and a 2nd water wash performed at the same ratio, vortexed, andcentrifuged at 4,400 rpm for 10 minutes. Following treatment, theemulsions were observed for the appearance of a visible oil layer.

4. Sonication

A sampling of conditions (samples #1, 9, 10, 14, 18, 19, 23, 27) fromthe chemical/heat treatment matrix were sonicated using a Misonix 3000sonicator equipped with a micro-tip on level 7. The suspension wassonicated for 3 minutes on a 30 sec on/20 sec off cycle. Aftertreatment, a 1.5 ml sample was centrifuged at 14 K rpm for 15 min andthe size of each of the resulting layers was measured.

5. Emulsion Freezing

Samples which generated emulsions were subjected to freezing at −20° C.for >24 hours. Following freezing, the samples were thawed at roomtemperature, vortexed, and centrifuged at 4,400 rpm for 10 minutes. Thesamples studied are listed in Table 22.

TABLE 22 Emulsions used in freezing cycle. Sample Sonicated 160 mN HCl,130° C. for 30 min Yes, 30 min cycle 120 mN HCl, 130° C. for 30 min No160 mN KOH 130° C. for 30 min Yes, 30 min cycle No chemical, 130° C. for30 min Yes, 30 min cycle No chemical, no heat Yes, 30 min cycle

Following treatment, the emulsions were observed for the appearance of avisible oil layer.

6. Enzyme Treatment Experiments #1

Several enzymes were evaluated as an alternative method of cellbreakage. These enzymes, all obtained from Sigma Aldrich, wereHemicellulase (P/N H2125), Pectinase (P/N P2401), Cellulase (C9422), andDriselase (D9515). 5 mL of cell material was prepared to various biomassconcentrations, and exchanged into buffer. The enzyme concentrationswere then prepared as described in Table 23.

TABLE 23 Enzyme treatment matrix. Enzyme conc. Incubation Incubation[BM] Enzyme (%) temp, ° C. time, hrs Incubation buffer g/L Hemicellulase1% 25° C. 12 100 mM Acetate, pH 4.6 150 Pectinase 1% 25° C. 12 100 mMAcetate, pH 4.6 150 Cellulase 1% 25° C. 12 100 mM Acetate, pH 4.6 150Driselase 1% 25° C. 12 100 mM Acetate, pH 4.6 150 Cocktail of all 4enzymes 1% of each 25° C. 12 100 mM Acetate, pH 4.6 150 Hemicellulase 1%37° C. 1 100 mM Acetate, pH 4.6 150 Pectinase 1% 37° C. 1 100 mMAcetate, pH 4.6 150 Cellulase 1% 37° C. 1 100 mM Acetate, pH 4.6 150Driselase 1% 37° C. 1 100 mM Acetate, pH 4.6 150 Cocktail of all 4enzymes 1% of each 37° C. 1 100 mM Acetate, pH 4.6 150Hemicellulase/Driselase 4%/2% 37° C. 12 50 mM Citrate, pH 4.5 150Hemicellulase/Driselase 4%/2% 50° C. 12 50 mM Citrate, pH 4.5 150Hemicellulase/Driselase 4%/2% 37° C. 12 50 mM Citrate, pH 4.5 100Hemicellulase/Driselase 4%/2% 50° C. 12 50 mM Citrate, pH 4.5 100Hemicellulase/Driselase 4%/2% 37° C. 12 50 mM Citrate, pH 4.5 50Hemicellulase/Driselase 4%/2% 50° C. 12 50 mM Citrate, pH 4.5 50Hemicellulase/Driselase 4%/2% 37° C. 12 50 mM Citrate, pH 4.5 25Hemicellulase/Driselase 4%/2% 50° C. 12 50 mM Citrate, pH 4.5 25

7. Enzyme Treatment Experiments #2

Additional enzymes were evaluated alone, and in combination with oneanother, and compared against disruption of cells by storing and agingthe cells with or without enzyme treatment. A polysaccharide-degradingenzyme mannose and a protease were evaluated for their ability todisrupt cells of Chlorella protothecoides. Also, the effect of storingand aging a sample of cultured cells on the weakening of the cellstructure to facilitate oil extraction was evaluated.

Three experiments were conducted. Chlorella protothecoides cells weregenerated via heterotrophic growth using glucose as the sole carbonsource. In experiments in which dried cells were used, fermentationbroth was centrifuged and the cell paste was subjected to drum drying.In experiments in which cells were stored for 7 days, storage was at 4°C. A polysaccharide-degrading enzyme (Mannaway 4.0 L) and protease(Alcalase 2.4 FG) (both from Novozymes) were used in the experiments, asindicated. Enzymes were added at a concentration of 0.2% weight/volume.

In the first experiment, dried cells were reconstituted in deionizedwater to a dry cell weight of 155 grams/liter in flasks, and maintainedat pH 7.5. Flasks were placed in an incubating shaker for 22 hours at50° C. Enzymatic cell lysis for sample 1 in Experiment 1 was veryeffective when combining enzymes, as evidenced by 90.4% of total oilbeing found in the emulsion and not in the pellet. Neither enzymeindividually was as effective as the combination of both, nor was thecontrol without enzyme treatment. The results are shown in Table 27below.

In the second experiment, fresh fermentation broth was separated intotwo aliquots for fresh and 7 day broth experiments. The fresh cells wereconcentrated by centrifugation and reconstituted in deionized water to170 g/l in flasks. Resuspended cell samples were then subdivided intotwo sets of 4 flasks, half of which were stored for 7 days at 4° C.before further treatment. For experimental treatment, 3 flasks containedcombinations of a protease and a polysaccharide-degrading mannase whilethe 4^(th) contained no enzymes (see Table 27 for experimentalconditions). Flasks were placed in an incubating shaker for 22 hours at50° C. This second experiment indicated extensive cell lysis with dualenzyme treatment at day 0 and day 7, as well as lysis in the absence ofenzymes after 7 days. The results are shown in Table 28 below.

In the third experiment, fermentation broth was collected from freshfermentations, concentrated by centrifugation and re-suspended indeionized water at two different concentrations of 71 and 115 g/l andagitated at 150 rpm in Applikon fermentors for 22 hours at 50° C. pH wasmaintained at 7.5 by feedback acid/base control. The results of thisthird experiment indicated that cell disruption and/or emulsionformation is facilitated by addition of enzymes and aging of the cells.The results are shown in Table 29 below.

8. Oil Analysis and Characterization

TLC was performed on aluminum backed silica TLC plates (Sigma#60805).The developing solvent (mobile phase) was hexane/diethyleter/acetic acid(80:20:1, by vol). Spots were visualized by spraying with 10% sulfuricacid and charring on a hotplate. Recovered oil samples from control,acidic, and basic conditions were analyzed to determine if anysignificant product degradation and/or modification occurred due to theprocessing conditions.

9. Results

(a) Heat and Chemical Treatment

The results from the chemical/heat treatment matrix are reported in FIG.3 and Table 24 below.

TABLE 24 Measurement of layers from chemical/heat treatment matrix.[KOH] [H₂SO₄] Temp, Pellet, Aqueous, Emulsion, Tube Condition mN mN 30min mm mm mm 1 Control 0 0 25° C. 15 15 0 2 40 mN KOH 40 0 25° C. 15 150 3 80 mN KOH 80 0 25° C. 15 15 0 4 120 mN KOH 120 0 25° C. 15 15 0 5160 mN KOH 160 0 25° C. 15 15 0 6 40 mN H₂SO₄ 0 40 25° C. 15 15 0 7 80mN H₂SO₄ 0 80 25° C. 15 15 0 8 120 mN 0 120 25° C. 15 15 0 H₂SO₄ 9 160mN 0 160 25° C. 15 15 0 H₂SO₄ 10 Control 0 0 65° C. 10 19 1 11 40 mN KOH40 0 65° C. 10 19 1 12 80 mN KOH 80 0 65° C. 10 19 1 13 120 mN KOH 120 065° C. 9 19 2 14 160 mN KOH 160 0 65° C. 8 19 3 15 40 mN H₂SO₄ 0 40 65°C. 10 19 1 16 80 mN H₂SO₄ 0 80 65° C. 10 19 1 17 120 mN 0 120 65° C. 1019 1 H₂SO₄ 18 160 mN 0 160 65° C. 10 19 1 H₂SO₄ 19 Control 0 0 65° C. 515 10 20 40 mN KOH 40 0 130° C.  10 18 2 21 80 mN KOH 80 0 130° C.  5 1510 22 120 mN KOH 120 0 130° C.  5 15 10 23 160 mN KOH 160 0 130° C.  515 10 24 40 mN H₂SO₄ 0 40 130° C.  2 23 5 25 80 mN H₂SO₄ 0 80 130° C.  119 10 26 120 mN 0 120 130° C.  1 19 10 H₂SO₄ 27 160 mN 0 160 130° C.  119 10 H₂SO₄

At 25° C., no difference was observed between the chemically treatedsamples and the control, and no emulsion formed.

At 65° C., an emulsion began to appear for the KOH treated samples,increasing in size with higher concentrations of caustic. The aqueousphase also became more turbid with increasing concentrations of KOH.There appeared to be a proportional decrease in the size of the pelletas the emulsion size increased. For the acidic treated samples at 65°C., a smaller emulsion formed relative to the caustic treated samples,and no increase in the size of the emulsion was observed with increasinglevel of acid, and the aqueous phase was clear and uncolored.

Samples heated to 130° C. all showed significantly larger emulsionscompared to 65° C., and comprised roughly ⅓ of the total sample volume.The aqueous phase of the caustic treated samples showed increasing darkcolor with increasing concentration of KOH.

In the acidic treated samples, the aqueous phase remained clear for allsamples. In contrast to the base treated materials, the pellet virtuallydisappeared for the acid treated preparations, indicating that the cellsand cell debris had partitioned to the emulsion and aqueous phase. Innone of the 27 conditions tested was a visible oil layer observed.

(b) Emulsion Water Washing

The emulsion of the caustic treated samples was poorly defined anddifficult to separate from the aqueous phase to prepare the emulsion forwater washing. In contrast, the acidic emulsion was firm and partitionedeasily from the aqueous phase. Under all washing conditions for the KOHtreated samples, the emulsion became thinner and thinner until it wasnearly gone. No visible oil layer was observed during either wash.

The acidic emulsions were easier to manipulate and separate from theaqueous phase. The emulsion size remained essentially unchanged duringboth washes. However, no visible oil layer was observed during eitherwash.

(c) Sonication

The results of the sonication are recorded in Table 25 below.

TABLE 25 Impact of sonication compared to corresponding pre-sonicatedsamples. [KOH] [H₂SO₄] Temp, Pellet, Emulsion, Tube mN mN 30 min mm mm 10 0 25° C. −5 +1 9 0 160 25° C. −5 +1 10 0 0 65° C. −5 +1 14 160 0 65°C. −3 +2 18 0 160 65° C. −5 +1 19 0 0 130° C. −2 −7 23 160 0 130° C. −3−7 27 0 160 130° C. +1 −7

The 25° C. and 65° C. sample emulsions increased in size while thepellet decreased relative to their pre-sonicated counterparts. For the130° C. samples, the emulsion decreased in size substantially, and thepellet of the control and base treated samples decreased, while thepellet increased for the acid treated preparation. There was some oillayer formation for the sonicated samples, particularly at the elevatedtemperatures.

(d) Temperature Reduction of Emulsion

The results of the emulsion freezing are recorded in Table 26 below.

TABLE 26 Results of emulsion freezing. Sample Sonicated Visible oillayer 160 mN HCl, 130° C. for 30 min Yes, 30 min cycle Yes 120 mN HCl,130° C. for 30 min No No 160 mN KOH 130° C. for 30 min Yes, 30 min cycleYes No chemical, 130° C. for 30 min Yes, 30 min cycle Yes No chemical,no heat Yes, 30 min cycle Yes

Reducing the temperature of the emulsion to −20° C. proved to be aneffective emulsion breaker. In all samples where the emulsion wassonicated, a large oil layer was observed. A representative result isshown in FIG. 4. When compared to the lipid content of the cell materialby HPLC, the oil layer constituted the majority of the oil from theemulsion.

(e) Enzymatic Treatment #1

Samples incubated at 25° C. and 37° C. at 1% enzyme levels showedminimal emulsion formation. Considerably larger emulsions formed at thehigher enzyme concentrations of 4%/2% Hemicellulase/Driselasecombination, with the 50° C. producing a larger emulsion compared to the37° C. sample. No oil layer was observed with enzyme treatment alone.Enzyme-generated emulsions subjected to sonication and then −20° C.,generated an oil layer.

(f) Enzymatic Treatment #2

Enzyme treatment combining a polysaccharide-degrading mannase and aprotease was found effective to disrupt cells from Chlorellaprotothecoides. Another technique found effective in weakening cellstructure enough to facilitate oil extraction was storing/aging thecells. Tables 27, 28 and 29 show the results of the three experimentsdescribed above, and FIG. 6 illustrates the resulting layers fromenzyme-treated (left tube) vs. untreated (right tube) biomass. In theuntreated tube, the cell pellet is considerably larger in size becauseit comprises a large lipid component. In neither case was lipid found inthe aqueous phase.

TABLE 27 Enzymatic treatment #2, experiment 1 results. Sample Number 1 23 4 ALCALASE 2.4 FG + + − − MANNAWAY 4.0 L + − + − % of oil in cellpellet 9.6% 30.7% 50.3% 97.8% % of oil in emulsion 90.4% 69.3% 49.7%2.2%

TABLE 28 Enzymatic treatment #2, experiment 2 results. Sample Number AGE1 2 3 4 ALCALASE 2.4 FG + + − − MANNAWAY 4.0 L + − + − DCW G/L 170.0170.0 170.0 170.0 DAYS W/W % W/W % W/W % W/W % % of oil in cell pellet 028.8% 48.2% 67.8% 100.0%  % of oil in emulsion 0 71.2% 51.8% 32.2%   0%% of oil in cell pellet 7 47.8% 81.3% 80.7% 81.8% % of oil in emulsion 752.2% 18.7% 19.3% 18.2%

TABLE 29 Enzymatic treatment #2, experiment 3 results. Sample Number 1 23 4 5 ALCALASE 2.4 FG − − + − − MANNAWAY 4.0 L − − + − − DCW G/L 71.7115.1 115.1 71.7 115.1 TEMPERATURE 50 50 50 50 50 PH 7.5 7.5 7.5 7.5 7.5W/W % W/W % W/W % W/W % W/W % % of oil in cell pellet 81.8% 63.0% 12.0%49.1% 42.0% % of oil in emulsion 18.2% 37.0% 88.0% 50.9% 58.0% age 0 0 07 7

(g) Oil Analysis and Characterization

An oil sample was isolated from each of the chemically treatedconditions and analyzed via TLC to determine if product degradation wasoccurring at the extreme pHs compared the control condition. TLCanalysis is shown in FIG. 5. The pattern showed a minor generation offree fatty acids (FFA's) under acidic conditions compared to the basetreated and control sample.

Example 8 Methods for Culturing Prototheca

Prototheca strains were cultivated to achieve a high percentage of oilby dry cell weight. Cryopreserved cells were thawed at room temperatureand 500 ul of cells were added to 4.5 ml of medium (4.2 g/L K₂HPO₄, 3.1g/L NaH₂PO₄, 0.24 g/L MgSO₄.7H₂O, 0.25 g/L Citric Acid monohydrate,0.025 g/L CaCl₂ 2H₂O, 2 g/L yeast extract) plus 2% glucose and grown for7 days at 28° C. with agitation (200 rpm) in a 6-well plate. Dry cellweights were determined by centrifuging 1 ml of culture at 14,000 rpmfor 5 min in a pre-weighed Eppendorf tube. The culture supernatant wasdiscarded and the resulting cell pellet washed with 1 ml of deionizedwater. The culture was again centrifuged, the supernatant discarded, andthe cell pellets placed at −80° C. until frozen. Samples were thenlyophilized for 24 hrs and dry cell weights calculated. Fordetermination of total lipid in cultures, 3 ml of culture was removedand subjected to analysis using an Ankom system (Ankom Inc., Macedon,N.Y.) according to the manufacturer's protocol. Samples were subjectedto solvent extraction with an Amkom XT10 extractor according to themanufacturer's protocol. Total lipid was determined as the difference inmass between acid hydrolyzed dried samples and solvent extracted, driedsamples. Percent oil dry cell weight measurements are shown in Table 30.

TABLE 30 Percent oil by dry cell weight Species Strain % Oil Protothecastagnora UTEX 327 13.14 Prototheca moriformis UTEX 1441 18.02 Protothecamoriformis UTEX 1435 27.17

Microalgae samples from the strains listed in Table 30, above, weregenotyped. Genomic DNA was isolated from algal biomass as follows. Cells(approximately 200 mg) were centifuged from liquid cultures 5 minutes at14,000×g. Cells were then resuspended in sterile distilled water,centrifuged 5 minutes at 14,000×g and the supernatant discarded. Asingle glass bead ˜2 mm in diameter was added to the biomass and tubeswere placed at −80° C. for at least 15 minutes. Samples were removed and150 μl of grinding buffer (1% Sarkosyl, 0.25 M Sucrose, 50 mM NaCl, 20mM EDTA, 100 mM Tris-HCl, pH 8.0, RNase A 0.5 ug/ul) was added. Pelletswere resuspended by vortexing briefly, followed by the addition of 40 ulof 5M NaCl. Samples were vortexed briefly, followed by the addition of66 μl of 5% CTAB (Cetyl trimethylammonium bromide) and a final briefvortex. Samples were next incubated at 65° C. for 10 minutes after whichthey were centrifuged at 14,000×g for 10 minutes. The supernatant wastransferred to a fresh tube and extracted once with 300 μl of Phenol:Chloroform:Isoamyl alcohol 12:12:1, followed by centrifugation for 5minutes at 14,000×g. The resulting aqueous phase was transferred to afresh tube containing 0.7 vol of isopropanol (˜190 μl), mixed byinversion and incubated at room temperature for 30 minutes or overnightat 4° C. DNA was recovered via centrifugation at 14,000×g for 10minutes. The resulting pellet was then washed twice with 70% ethanol,followed by a final wash with 100% ethanol. Pellets were air dried for20-30 minutes at room temperature followed by resuspension in 50 μl of10 mM TrisCl, 1 mM EDTA (pH 8.0).

Five μl of total algal DNA, prepared as described above, was diluted1:50 in 10 mM Tris, pH 8.0. PCR reactions, final volume 20 μl, were setup as follows. Ten μl of 2× iProof HF master mix (BIO-RAD) was added to0.4 μl primer SZ02613 (5′-TGTTGAAGAATGAGCCGGCGAC-3′ (SEQ ID NO:5) at 10mM stock concentration). This primer sequence runs from position 567-588in Gen Bank accession no. L43357 and is highly conserved in higherplants and algal plastid genomes. This was followed by the addition of0.4 μl primer SZ02615 (5′-CAGTGAGCTATTACGCACTC-3′ (SEQ ID NO:6) at 10 mMstock concentration). This primer sequence is complementary to position1112-1093 in Gen Bank accession no. L43357 and is highly conserved inhigher plants and algal plastid genomes. Next, 5 μl of diluted total DNAand 3.2 μl dH₂O were added. PCR reactions were run as follows: 98° C.,45″; 98° C., 8″; 53° C., 12″; 72° C., 20″ for 35 cycles followed by 72°C. for 1 mM and holding at 25° C. For purification of PCR products, 20μl of 10 mM Tris, pH 8.0, was added to each reaction, followed byextraction with 40 μl of Phenol:Chloroform:isoamyl alcohol 12:12:1,vortexing and centrifuging at 14,000×g for 5 minutes. PCR reactions wereapplied to S-400 columns (GE Healthcare) and centrifuged for 2 minutesat 3,000×g. Purified PCR products were subsequently TOPO cloned intoPCR8/GW/TOPO and positive clones selected for on LB/Spec plates.Purified plasmid DNA was sequenced in both directions using M13 forwardand reverse primers. In total, twelve Prototheca strains were selectedto have their 23S rRNA DNA sequenced and the sequences are listed in theSequence Listing. A summary of the strains and Sequence Listing Numbersis included below in Table 31. The sequences were analyzed for overalldivergence from the UTEX 1435 (SEQ ID NO: 17) sequence. Two pairsemerged (UTEX 329/UTEX 1533 and UTEX 329/UTEX 1440) as the mostdivergent. In both cases, pairwise alignment resulted in 75.0% pairwisesequence identity. The percent sequence identity to UTEX 1435 is alsoincluded below in Table 31.

TABLE 31 Genotyped Prototheca strains. % nt Species Strain identity SEQID NO. Prototheca kruegani UTEX 329 75.2 SEQ ID NO: 34 Protothecawickerhamii UTEX 1440 99 SEQ ID NO: 35 Prototheca stagnora UTEX 144275.7 SEQ ID NO: 16 Prototheca moriformis UTEX 288 75.4 SEQ ID NO: 36Prototheca moriformis UTEX 1439; 100 SEQ ID NO: 17 1441; 1435; 1437Prototheca wikerhamii UTEX 1533 99.8 SEQ ID NO: 37 Prototheca moriformisUTEX 1434 75.9 SEQ ID NO: 38 Prototheca zopfii UTEX 1438 75.7 SEQ ID NO:39 Prototheca moriformis UTEX 1436 88.9 SEQ ID NO: 32

Lipid samples from a subset of the above-listed strains were analyzedfor lipid profile using HPLC. Results are shown below in Table 32.

TABLE 32 Diversity of lipid chains in Prototheca species. Strain C14:0C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 UTEX 0 12.01 0 0 50.3317.14 0 0 0 327 UTEX 1.41 29.44 0.70 3.05 57.72 12.37 0.97 0.33 0 1441UTEX 1.09 25.77 0 2.75 54.01 11.90 2.44 0 0 1435

Algal plastid transit peptides were identified through the analysis ofUTEX 1435 (Prototheca moriformis) or UTEX 250 (Chlorella protothecoides)cDNA libraries. cDNAs encoding potentially plastid targeted proteinsbased upon BLAST hit homology to other known plastid targeted proteinswere subjected to further analysis by the sowftawre programs PSORT(http://psort.ims.u-tokyo.ac.jp/form.html), ChloroP(http://www.cbs.dtu.dk/services/ChloroP/) are TargetP(http://www.cbs.dtu.dk/services/TargetP/). Candidate plastid transitpeptides identified through at least one of these three programs werethen PCR amplified from the appropriate genomic DNA. Below, in Table 33,is a summary of the algal plastid targeting amino acid sequences (PTS)that were identified from this screen. Also included are the amino acidsequences of plant fatty acyl-ACP thioesterases that are used in theheterologous expression Examples below.

TABLE 33 Summary of algal plastic targeting amino acid sequences. cDNASEQ ID NO. P. moriformis isopentenyl diphosphate synthase PTS SEQ ID NO:40 P. moriformis delta 12 fatty acid desaturase PTS SEQ ID NO: 41 P.moriformis stearoyl ACP desaturase PTS SEQ ID NO: 42 C. protothecoidesstearoyl ACP desaturase PTS SEQ ID NO: 43 Cuphea hookeriana fattyacyl-ACP thioesterase SEQ ID NO: 44 (C8-10) Umbellularia californicafatty acyl-ACP thioesterase SEQ ID NO: 45 (C12) Cinnamomum camphorafatty acyl-ACP thioesterase SEQ ID NO: 46 (C14)

Example 9 Methods for Transforming Prototheca

1. General Method for Biolistic transformation of Prototheca

S550d gold carriers from Seashell Technology were prepared according tothe protocol from manufacturer. Linearized plasmid (20 μg) was mixedwith 50 μl of binding buffer and 60 μl (30 mg) of S550d gold carriersand incubated in ice for 1 min Precipitation buffer (100 μl) was added,and the mixture was incubated in ice for another 1 min. After vortexing,DNA-coated particles were pelleted by spinning at 10,000 rpm in anEppendorf 5415C microfuge for 10 seconds. The gold pellet was washedonce with 500 μl of cold 100% ethanol, pelleted by brief spinning in themicrofuge, and resuspended with 50 μl of ice-cold ethanol. After a brief(1-2 sec) sonication, 10 μl of DNA-coated particles were immediatelytransferred to the carrier membrane.

Prototheca strains were grown in proteose medium (2 g/L yeast extract,2.94 mM NaNO3, 0.17 mM CaCl2.2H₂O, 0.3 mM MgSO4.7H₂O, 0.4 mM K2HPO4,1.28 mM KH2PO4, 0.43 mM NaCl) on a gyratory shaker until it reaches acell density of 2×10⁶ cells/ml. The cells were harvested, washed oncewith sterile distilled water, and resuspended in 50 μl of medium. 1×10⁷cells were spread in the center third of a non-selective proteose mediaplate. The cells were bombarded with the PDS-1000/He Biolistic ParticleDelivery system (Bio-Rad). Rupture disks (1100 and 1350 psi) were used,and the plates are placed 9 and 12 cm below the screen/macrocarrierassembly. The cells were allowed to recover at 25° C. for 12-24 h. Uponrecovery, the cells were scraped from the plates with a rubber spatula,mixed with 100 μl of medium and spread on plates containing theappropriate antibiotic selection. After 7-10 days of incubation at 25°C., colonies representing transformed cells were visible on the platesfrom 1100 and 1350 psi rupture discs and from 9 and 12 cm distances.Colonies were picked and spotted on selective agar plates for a secondround of selection.

2. Transformation of Prototheca with G418 Resistance Gene

Prototheca moriformis and other Prototheca strains sensitive to G418 canbe transformed using the methods described below. G418 is anaminoglycoside antibiotic that inhibits the function of 80S ribosomesand thereby inhibits protein synthesis. The corresponding resistancegene functions through phosphorylation, resulting in inactivation ofG418. Prototheca strains UTEX 1435, UTEX 1439 and UTEX 1437 wereselected for transformation. All three Prototheca strains were genotypedusing the methods described above. All three Prototheca strains hadidentical 23s rRNA genomic sequences (SEQ ID NO:17).

All transformation cassettes were cloned as EcoRI-SacI fragments intopUC19. Standard molecular biology techniques were used in theconstruction of all vectors according to Sambrook and Russell, 2001. TheC. reinhardtii beta-tubulin promoter/5′UTR was obtained from plasmidpHyg3 (Berthold et al., (2002) Protist: 153(4), pp 401-412) by PCR as anEcoRI-AscI fragment. The Chlorella vulgaris nitrate reductase 3′UTR wasobtained from genomic DNA isolated from UTEX strain 1803 via PCR usingthe following primer pairs:

Forward: (SEQ ID NO: 47) 5′TGACCTAGGTGATTAATTAACTCGAGGCAGCAGCAGCTCGGATAGTA TCG 3′ Reverse:(SEQ ID NO: 48) 5′ CTACGAGCTCAAGCTTTCCATTTGTGTTC CCATCCCACTACTTCC 3′

The Chlorella sorokiniana glutamate dehydrogenase promoter/UTR wasobtained via PCR of genomic DNA isolated from UTEX strain 1230 via PCRusing the following primer pairs:

Forward: (SEQ ID NO: 34) 5′ GATCAGAATTCCGCCTGCAACGCAAGG GCAGC 3′Reverse: (SEQ ID NO: 35) 5′ GCATACTAGTGGCGGGACGGAGAGA GGGCG 3′

Codon optimization was based on the codons for Prototheca moriformis.The sequence of the non-codon optimized neomycin phosphotransferase(nptII) cassette was synthesized as an AscI-XhoI fragment and was basedon upon the sequence of Genbank Accession No. YP_(—)788126. The codonoptimized nptII cassette was also based on this Genbank Accessionnumber.

The three Prototheca strains were transformed using biolistic methodsdescribed above. Briefly, the Prototheca strains were grownheterophically in liquid medium containing 2% glucose until they reachedthe desired cell density (1×10⁷ cells/mL to 5×10⁷ cells/mL). The cellswere harvested, washed once with sterile distilled water and resuspendedat 1×10⁸ cells/mL. 0.5 mL of cells were then spread out on anon-selective solid media plate and allowed to dry in a sterile hood.The cells were bombarded with the PDS-1000/He Biolistic ParticleDelivery System (BioRad). The cells were allowed to recover at 25° C.for 24 hours. Upon recovery, the cells were removed by washing plateswith 1 mL of sterile media and transferring to fresh plates containing100 μg/mL G418. Cells were allowed to dry in a sterile hood and colonieswere allowed to form on the plate at room temperature for up to threeweeks. Colonies of UTEX 1435, UTEX 1439 and UTEX 1437 were picked andspotted on selective agar plates for a second round of selection.

A subset of colonies that survived a second round of selection describedabove, were cultured in small volume and genomic DNA and RNA wereextracted using standard molecular biology methods. Southern blots weredone on genomic DNA extracted from untransformed (WT), the transformantsand plasmid DNA. DNA from each sample was run on 0.8% agarose gels afterthe following treatments: undigested (U), digested with AvrII (A),digested with NcoI (N), digested with SacI (S). DNA from these gels wasblotted on Nylon+membranes (Amersham). These membranes were probed witha fragment corresponding to the entire coding region of the nptII gene(NeoR probe). FIG. 7 shows maps of the cassettes used in thetransformations. FIG. 8 shows the results of Southern blot analysis onthree transformants (all generated in UTEX strain 1435) (1, 2, and 3)transformed with either the beta-tubulin::neo::nit (SEQ ID NO: 49)(transformants 1 and 2) or glutamate dehydrogenase:neo:nit (SEQ ID NO:50) (transformant 3). The glutamate dehydrogenase:neo:nit transformingplasmid was run as a control and cut with both NcoI and SacI. AvrII doesnot cut in this plasmid. Genomic DNA isolated from untransformed UTEXstrain 1435 shows no hybridization to the NeoR probe.

Additional transformants containing the codon-optimized glutamatedehydrogenase:neo:nit (SEQ ID NO: 51) and codon-optimizedβ-tubulin::neo::nit (SEQ ID NO:52) constructs were picked and analyzedby Southern blot analysis. As expected, only digests with Sad showlinearization of the transforming DNA. These transformation events areconsistent with integration events that occur in the form of oligomersof the transforming plasmid. Only upon digestion with restrictionenzymes that cut within the transforming plasmid DNA do these moleculescollapse down the size of the transforming plasmid.

Southern blot analysis was also performed on transformants generatedupon transformation of Prototheca strains UTEX 1437 and UTEX 1439 withthe glutamate dehydrogenase::neo::nit cassette. The blot was probed withthe NeoR probe probe and the results are similar to the UTEX 1435transformants. The results are indicative of integration eventscharacterized by oligomerization and integration of the transformingplasmid. This type of integration event is known to occur quite commonlyin Dictyostelium discoideum (see, for example, Kuspa, A. and Loomis, W.(1992) PNAS, 89:8803-8807 and Morio et al., (1995) J. Plant Res.108:111-114).

To further confirm expression of the transforming plasmid, Northern blotanalysis and RT-PCR analysis were performed on selected transformants.RNA extraction was performed using Trizol Reagent according tomanufacturer's instructions. Northern blot analysis were run accordingto methods published in Sambrook and Russel, 2001. Total RNA (15 μg)isolated from five UTEX 1435 transformants and untransformed UTEX 1435(control lanes) was separated on 1% agarose-formaldehyde gel and blottedon nylon membrane. The blot was hybridized to the neo-non-optimizedprobe specific for transgene sequences in transformants 1 and 3. The twoother transformants RNAs express the codon-optimized version of theneo-transgene and, as expected, based on the sequence homology betweenthe optimized and non-optimized neo genes, showed significantly lowerhybridization signal.

RNA (1 μg) was extracted from untransformed Prototheca strain UTEX 1435and two representative UTEX 1435 transformants and reverse transcribedusing an oligio dT primer or a gene specific primer. Subsequently thesecDNAs (in duplicate) were subjected to qPCR analysis on ABI VeritiThermocycler using SYBR-Green qPCR chemistry using the following primers(nptII):

(SEQ ID NO: 53) Forward: 5′ GCCGCGACTGGCTGCTGCTGG 3′ (SEQ ID NO: 54)Reverse: 5′ AGGTCCTCGCCGTCGGGCATG 3′

Possible genomic DNA contamination was ruled out by a no reversetranscriptase negative control sample. The results indicated that theNeoR genes used to transform these strains is actively transcribed inthe transformants.

3. Transformation of Prototheca with Secreted Heterologous SucroseInvertase

All of the following experiments were performed using liquid medium/agarplates based on the basal medium described in Ueno et al., (2002) JBioscience and Bioengineering 94(2):160-65, with the addition of traceminerals described in U.S. Pat. Nos. 5,900,370, and 1×DAS VitaminCocktail (1000× solution): tricine: 9 g, thiamine HCL: 0.67 g, biotin:0.01 g, cyannocobalamin (vitamin B12): 0.008 g, calcium pantothenate:0.02 g and p-aminobenzoic acid: 0.04 g).

Two plasmid constructs were assembled using standard recombinant DNAtechniques. The yeast sucrose invertase genes (one codon optimized andone non-codon optimized), suc2, were under the control of the Chlorellareinhardtii beta-tubulin promoter/5′UTR and had the Chlorella vulgarisnitrate reductase 3′UTR. The sequences (including the 5′UTR and 3′UTRsequences) for the non-codon optimized (Crβ-tub::NCO-suc2::CvNitRed)construct, SEQ ID NO: 55, and codon optimized(Crβ-tub::CO-suc2::CvNitRed) construct, SEQ ID NO: 56, are listed in theSequence Listing. Codon optimization was based on Prototheca sp (seeTable 4). FIG. 9 shows a schematic of the two constructs with therelevant restriction cloning sites and arrows indicating the directionof transcription. Selection was provided by Neo R. Codon optimizationwas based on preferred codon usage in Prototheca strains in Table 4.

Preparation of the DNA/gold microcarrier: DNA/gold microcarriers wereprepared immediately before use and stored on ice until applied tomacrocarriers. The plasmid DNA (in TE buffer) was added to 50 μl ofbinding buffer. Saturation of the gold beads was achieved at 15 μgplasmid DNA for 3 mg gold carrier. The binding buffer and DNA were mixedwell via vortexing. The DNA and binding buffer should be pre-mix priorto gold addition to ensure uniformed plasmid binding to gold carrierparticles. 60 μl of S550d (Seashell Technologies, San Diego, Calif.)gold carrier was added to the DNA/binding buffer mixture. For a goldstock at 50 mg/ml, addition of 60 μA results in an optimal ratio of 15μg DNA/3 mg gold carrier. The gold carrier/DNA mixture was allowed toincubate on ice for 1 minute and then 100 μl of precipitation buffer wasadded. The mixture was allowed to incubate again on ice for 1 minute andthen briefly vortexed and centrifuged at 10,000 rpm at room temperaturefor 10 seconds to pellet the gold carrier. The supernatant was carefullyremoved with a pipette and the pellet was washed with 500 μl of ice cold100% ethanol. The gold particles were re-pelleted by centrifuging againat 10,000 rpm for 10 seconds. The ethanol was removed and 50 μl of icecold ethanol was added to the gold mixture. Immediately prior toapplying the gold to macrocarriers, the gold/ethanol was resuspendedwith a brief 1-2 second pulse at level 2 on a MISONIX sonicator usingthe micro tip. Immediately after resuspension, 10 μl of the dispersedgold particles was transferred to the macrocarrier and allowed to dry ina sterile hood.

The two Prototheca moriformis strains (UTEX 1435 and 1441) were grownheterotrophically in liquid medium containing 2% glucose fromcryopreserved vials. Each strain was grown to a density of 10⁷ cells/ml.This seed culture was then diluted with fresh media to a density of 10⁵cells/ml and allowed to grow for 12-15 hours to achieve a final celldensity of approximately 10⁶ cells/ml. The microalgae were aliquotedinto 50 ml conical tubes and centrifuged for 10 minutes at 3500 rpm. Thecells were washed with fresh medium and centrifuged again for 10 minutesat 3500 rpm. The cells were then resuspended at a density of 1.25×10⁸cells/ml in fresh medium.

In a sterile hood, 0.4 ml of the above-prepared cells were removed andplaced directly in the center of an agar plate (without selectionagent). The plate was gently swirled with a level circular motion toevenly distribute the cells to a diameter of no more than 3 cm. Thecells were allowed to dry onto the plates in the sterile hood forapproximately 30-40 minutes and then were bombarded at a rupture diskpressure of 1350 psi and a plate to macrocarrier distance of 6 cm. Theplates were then covered and wrapped with parafilm and allowed toincubate under low light for 24 hours.

After the 24 hour recovery, 1 ml of sterile medium (with no glucose) wasadded to the lawn of cells. The cells were resuspended using a sterileloop, applied in a circular motion to the lawn of cells and theresuspended cells were collected using a sterile pipette. The cells werethen plated onto a fresh agar plate with 2% glucose and 100 μg/ml G418.The appearance of colonies occurred 7-12 days after plating. Individualcolonies were picked and grown in selective medium with 2% glucose and100 μg/ml G418. The wildtype (untransformed) and transgenic cells werethen analyzed for successful introduction, integration and expression ofthe transgene.

Genomic DNA from transformed Prototheca moriformis UTEX 1435 and 1441and their wildtype (untransformed) counterparts were isolated usingstandard methods. Briefly, the cells were centrifuged for 5 minutes at14,000 rpm in a standard table top Eppendorf centrifuge (model 5418) andflash frozen prior to DNA extraction. Cell pellets were lysed by adding200 uL of Lysis buffer (100 mM Tris HCl, pH 8.0, 1% Lauryl Sarcosine, 50mM NaCl, 20 mM EDTA, 0.25 M sucrose, 0.5 mg/ml RNase A) for every100-200 mg of cells (wet weight) and vortexing for 30-60 seconds. Cetyltrimethyammonium bromide (CTAB) and NaCl were brought to 1% and 1 M,respectively, and cell extracts were incubated at 60-65° C. for 10minutes. Subsequently, extracts were clarified via centrifugation at14,000 rpm for 10 minutes and the resulting supernatant was extractedwith an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1).Samples were then centrifuged for 5 minutes at 14,000 rpm and theaqueous phase removed. DNA was precipitated with 0.7 volumes ofisopropanol. DNA was pelleted via centrifugation at 14,000 rpm for 10minutes and washed twice with 80% ethanol, and once with ethanol. Afterdrying, DNA was resuspended in 10 mM Tris HCl, pH 8.0 and DNAconcentrations were determined by using PicoGreen fluorescencequantification assay (Molecular Probes).

RNA from transformed Prototheca moriformis UTEX 1435 and 1441 and theirwildtype (untransformed) counterparts were isolated using standardmethods. Briefly, the cells were centrifuged for 5 minutes at 14,000 rpmin a standard table top Eppendorf centrifuge (model 5418) and flashfrozen before RNA extraction. Cell pellets were lysed by addition of 1mL of Trizol reagent (Sigma) for every 100 mg of cells (wet weight) andby vortexing for 1-2 minutes. Samples were incubated at room temperaturefor 5 minutes and subsequently adjusted with 200 uL of chloroform per 1mL of Trizol reagent. After extensive shaking, cells were incubated atroom temperature for 15 minutes and then subjected to centrifugation at14000 rpm for 15 minutes in a refrigerated table top microcentrifuge.RNA partitioning to the upper aqueous phase was removed and precipitatedby addition of isopropanol (500 uL per 1 ml of Trizol reagent). RNA wascollected by centrifugation for 10 minutes and the resulting pelletwashed twice with 1 mL of 80% ethanol, dried, and resuspended in RNAsefree water. RNA concentration was estimated by RiboGreen fluorescencequantification assay (Molecular Probes).

Expression of neomycin phosphotransferase gene conferring G418antibiotic resistance and yeast invertase was assayed in non-transformedPrototheca moriformis UTEX 1435 and 1441 and transformants T98 (UTEX1435 transformant) and T97 (UTEX 1441 transformant) using reversetranscription quantitative PCR analysis (RT-qPCR). 20 ng total RNA(isolated as described above) was subjected to one step RT-qPCR analysisusing iScript SYBR Green RT-PCR kit (BioRad Laboratories) and primerpairs targeting the neomycin resistance gene (forward primer 5′CCGCCGTGCTGGACGTGGTG 3′ and reverse primer 5′ GGTGGCGGGGTCCAGGGTGT 3′;SEQ ID NOs: 57 and 58, respectively) and suc2 invertase transcripts(forward primer 5′ CGGCCGGCGGCTCCTTCAAC 3′ and reverse primer 5′GGCGCTCCCGTAGGTCGGGT 3′; SEQ ID NO: 59 and 60, respectively). Endogenousbeta-tubulin transcripts served as an internal positive control for PCRamplification and as a normalization reference to estimate relativetranscript levels.

Both codon optimized and non-codon optimized constructs were transformedinto UTEX 1435 and 1441 Prototheca moriformis cells as described above.Initially, transformants were obtained with both constructs and thepresence of the transgene was verified by Southern blot analysisfollowed by RTPCR to confirm the presence of the DNA and mRNA from thetransgene. For the Southern blot analysis, genomic DNA isolated asdescribed above was electrophoresed on 0.7% agarose gels in 1×TAEbuffer. Cells were processed as described in Sambrook et al. (MolecularCloning; A Laboratory Manual, 2^(nd) Edition. Cold Spring HarborLaboratory Press, 1989). Probes were prepared by random priming andhybridizations carried out as described in Sambrook et al. Transformantsfrom both the codon optimized and the non-codon optimized constructsshowed the presence of the invertase cassette, while the non-transformedcontrol was negative. Invertase mRNA was also detected in transformantswith both the codon optimized and non-codon optimized constructs.

To confirm that the transformants were expressing an active invertaseprotein, the transformants were plated on sucrose plates. Thetransformants containing the non-codon optimized cassette failed to growon the sucrose containing plates, indicating that, while the gene andthe mRNA encoding the SUC2 protein were present, the protein was either(1) not being translated, or (2) being translated, but not accumulatingto levels sufficient to allow for growth on sucrose as the sole carbonsource. The transformants with the codon optimized cassette grew on thesucrose containing plates. To assess the levels of invertase beingexpressed by these transformants, two clones (T98 and T97) weresubjected to an invertase assay of whole cells scraped from solid mediumand direct sampling and quantitation of sugars in the culturesupernatants after 48 hours of growth in liquid medium containing 2%sucrose as the sole carbon source.

For the invertase assay, the cells (T98 and T97) were grown on platescontaining 2% sucrose, scraped off and assayed for invertase activity.10 μl of the scraped cells was mixed with 40 μl of 50 mM NaOAc pH 5.1.12.5 μl of 0.5M sucrose was added to the cell mixture and incubated at37° C. for 10-30 minutes. To stop the reaction, 75 μl of 0.2M K₂HPO₄ wasadded. To assay for glucose liberated, 500 μl of reconstituted reagent(glucose oxidase/peroxidase+o-Dianisidine) from Sigma (GAGO-20 assaykit) was added to each tube and incubated at 37° C. for 30 minutes. Aglucose standard curve was also created at this time (range: 25 μg to0.3 μg glucose). After incubation, 500 μl of 6N HCl was added to stopthe reaction and to develop the color. The samples were read at 540 nm.The amount of glucose liberated was calculated from the glucose standardcurve using the formula y=m×+c, where y is the 540 nm reading, and x isμg of glucose. Weight of glucose was converted to moles of glucose, andgiven the equimolar relationship between moles of sucrose hydrolyzed tomoles of glucose generated, the data was expressed as nmoles of sucrosehydrolyzed per unit time. The assay showed that both T98 and T97 cloneswere able to hydrolyze sucrose, indicating that a functional sucroseinvertase was being produced and secreted by the cells.

For the sugar analysis on liquid culture media after 48 hours of algalgrowth, T97 and T98 cells were grown in 2% sucrose containing medium for48 hours and the culture media were processed for sugar analysis.Culture broths from each transformant (and negative non-transformed cellcontrol) were centrifuged at 14,000 rpm for 5 minutes. The resultingsupernatant was removed and subjected to HPLC/ELSD (evaporative lightscattering detection). The amount of sugar in each sample was determinedusing external standards and liner regression analysis. The sucroselevels in the culture media of the transformants were very low (lessthan 1.2 g/L, and in most cases 0 g/L). In the negative controls, thesucrose levels remained high, at approximately 19 g/L after 48 hours ofgrowth.

These results were consistant with the invertase activity results, andtaken together, indicated that the codon optimized transformants, T97and T98, secreted an active sucrose invertase that allowed themicroalgae to utilize sucrose as the sole carbon source in contrast to(1) the non-codon optimized transformants and (2) the non-transformedwildtype microalgae, both of which could not utilize sucrose as the solecarbon source in the culture medium.

Prototheca moriformis strains, T98 and T97, expressing a functional,secreted sucrose invertase (SUC2) transgene were assayed for growth andlipid production using sucrose as the sole carbon source.

Wild type (untransformed), T98 and T97 strains were grown in growthmedia (as described above) containing either 4% glucose or 4% sucrose asthe sole carbon source under heterotrophic conditions for approximately6 days. Growth, as determined by A750 optical density readings weretaken of all four samples every 24 hours and the dry cell weight of thecultures and lipid profiles were determined after the 6 days of growth.The optical density readings of the transgenic strains grown in both theglucose and sucrose conditions were comparable to the wildtype strainsgrown in the glucose conditions. These results indicate that thetransgenic strains were able to grow on either glucose or sucrose as thesole carbon source at a rate equal to wildtype strains in glucoseconditions. The non-transformed, wildtype strains did not grow in thesucrose-only condition.

The biomass for the wildtype strain grown on glucose and T98 straingrown on sucrose was analyzed for lipid profile. Lipid samples wereprepared from dried biomass (lyophilized) using an Acid HydrolysisSystem (Ankom Technology, NY) according to manufacturer's instructions.Lipid profile determinations were carried as described in Example 6. Thelipid profile for the non-transformed Prototheca moriformis UTEX 1435strain, grown on glucose as the sole carbon source and two colonal T98strains (UTEX 1435 transformed with a sucrose invertase transgene),grown on sucrose as the sole carbon source, are disclosed in Table 34(wildtype UTEX 1435 and T98 clone 8 and clone 11 below. C:19:0 lipid wasused as an internal calibration control.

TABLE 34 Lipid profile of wildtype UTEX 1435 and UTEX 1435 clones withsuc2 transgene. wildtype T98 clone 11 T98 clone 8 Name (Area % - ISTD)(Area % - ISTD) (Area % - ISTD) C 12:0 0.05 0.05 0.05 C 14:0 1.66 1.511.48 C 14:1 0.04 nd nd C 15:0 0.05 0.05 0.04 C 16:0 27.27 26.39  26.50 C 16:1 0.86 0.80 0.84 C 17:0 0.15 0.18 0.14 C 17:1 0.05 0.07 0.05 C 18:03.35 4.37 4.50 C 18:1 53.05 54.48  54.50  C 18:2 11.79 10.33  10.24  C19:0 (ISTD) — — — C 18:3 alpha 0.90 0.84 0.81 C 20:0 0.32 0.40 0.38 C20:1 0.10 0.13 0.12 C 20:1 0.04 0.05 0.04 C 22:0 0.12 0.16 0.12 C 20:30.07 0.08 0.07 C 24:0 0.12 0.11 0.10

Oil extracted from wildtype Prototheca moriformis UTEX 1435 (via solventextraction or using an expeller press) was analyzed for carotenoids,chlorophyll, tocopherols, other sterols and tocotrienols. The resultsare summarized below in Table 35.

TABLE 35 Carotenoid, chlorophyll, tocopherol/sterols and tocotrienolanalysis in oil extracted from Prototheca moriformis (UTEX 1435).Pressed oil Solvent extracted oil (mcg/ml) (mcg/ml) cis-Lutein 0.0410.042 trans-Lutein 0.140 0.112 trans-Zeaxanthin 0.045 0.039cis-Zeaxanthin 0.007 0.013 t-alpha-Crytoxanthin 0.007 0.010t-beta-Crytoxanthin 0.009 0.010 t-alpha-Carotene 0.003 0.001c-alpha-Carotene none detected none detected t-beta-Carotene 0.010 0.0099-cis-beta-Carotene 0.004 0.002 Lycopene none detected none detectedTotal Carotenoids 0.267 0.238 Chlorophyll <0.01 mg/kg <0.01 mg/kgTocopherols and Sterols Pressed oil Solvent extracted oil (mg/100 g)(mg/100 g) gamma Tocopherol 0.49 0.49 Campesterol 6.09 6.05 Stigmasterol47.6 47.8 Beta-sitosterol 11.6 11.5 Other sterols 445 446 TocotrienolsPressed oil Solvent extracted oil (mg/g) (mg/g) alpha Tocotrienol 0.260.26 beta Tocotrienol <0.01 <0.01 gamma Tocotrienol 0.10 0.10 detalTocotrienol <0.01 <0.01 Total Tocotrienols 0.36 0.36

The ability of using sucrose as the sole carbon source as the selectionfactor for clones containing the suc2 transgene construct instead ofG418 (or another antibiotic) was assessed using the positive suc2 genetransformants. A subset of the positive transformants was grown onplates containing sucrose as the sole carbon source and withoutantibiotic selection for 24 doublings. The clones were then challengedwith plates containing glucose as the sole carbon source and G418. Therewas a subset of clones that did not grow on the glucose+G418 condition,indicating a loss of expression of the transgene. An additionalexperiment was performed using a plate containing sucrose as the solecarbon source and no G418 and streaking out a suc2 transgene expressingclone on one half of the plate and wild-type Prototheca moriformis onthe other half of the plate. Growth was seen with both the wild-type andtransgene-containing Prototheca moriformis cells. Wild-type Protothecamoriformis has not demonstrated the ability to grow on sucrose,therefore, this result shows that unlike antibiotic resistance, the useof sucrose/invertase selection is not cell-autonomous. It is very likelythat the transformants were secreting enough sucrose invertase into theplate/media to support wildtype growth as the sucrose was hydrolyzedinto fructose and glucose.

Example 10 Recombinant Prototheca with Exogenous TE Gene

As described above, Prototheca strains can be transformed with exogenousgenes. Prototheca moriformis (UTEX 1435) was transformed, using methodsdescribed above, with either Umbellularia californica C12 thioesterasegene or Cinnamomum camphora C14 thiotesterase gene (both codon optimizedaccording to Table 4). Each of the transformation constructs contained aChlorella sorokiniana glutamate dehydrogenase promoter/5′UTR region (SEQID NO: 61) to drive expression of the thioesterase transgene. Thethioesterase transgenes coding regions of Umbellularia californica C12thioesterase (SEQ ID NO: 62) or Cinnamomum camphora C14 thioesterase(SEQ ID NO: 63), each with the native putative plastid targetingsequence. Immediately following the thioesterase coding sequence is thecoding sequence for a c-terminal 3×-FLAG tag (SEQ ID NO: 64), followedby the Chlorella vulgaris nitrate reductase 3′UTR (SEQ ID NO: 65).

Preparation of the DNA, gold microcarrier and Prototheca moriformis(UTEX 1435) cells were performed using the methods described above inExample 9. The microalgae were bombarded using the gold microcarrier—DNAmixture and plated on selection plates containing 2% glucose and 100μg/ml G418. The colonies were allowed to develop for 7 to 12 days andcolonies were picked from each transformation plate and screened for DNAconstruct incorporation using Southern blots assays and expression ofthe thioesterase constructs were screened using RT-PCR.

Positive clones were picked from both the C12 and C14 thioesterasetransformation plates and screened for construct incorporation usingSouthern blot assays. Southern blot assays were carried out usingstandard methods (and described above in Example 9) using an optimized cprobes, based on the sequence in SEQ ID NO: 62 and SEQ ID NO: 63.Transforming plasmid DNA was run as a positive control. Out of theclones that were positive for construct incorporation, a subset wasselected for reverse transcription quantitative PCR(RT-qPCR) analysisfor C12 thioesterase and C14 thioesterase expression.

RNA isolation was performed using methods described in Example 9 aboveand RT-qPCR of the positive clones were performed using 20 ng of totalRNA from each clone using the below-described primer pair and iScriptSYBR Green RT-PCR kit (Bio-Rad Laboratories) according to manufacturer'sprotocol. Wildtype (non-transformed) Prototheca moriformis total RNA wasincluded as a negative control. mRNA expression was expressed asrelative fold expression (RFE) as compared to negative control. Theprimers that were used in the C12 thioesterase transformation RT-qPCRscreening were: U. californica C12 thioesterase PCR primers:

(SEQ ID NO: 66) Forward: 5′ CTGGGCGACGGCTTCGGCAC 3′ (SEQ ID NO: 67)Reverse: 5′ AAGTCGCGGCGCATGCCGTT 3′

The primers that were used in the C14 thioesterase transformationRT-qPCR screening were:

Cinnamomum camphora C14 thioesterase PCR primers:

(SEQ ID NO: 68) Forward: 5′ TACCCCGCCTGGGGCGACAC 3′ (SEQ ID NO: 69)Reverse: 5′ CTTGCTCAGGCGGCGGGTGC 3′

RT-qPCR results for C12 thioesterase expression in the positive clonesshowed an increased RFE of about 40 fold to over 2000 fold increasedexpression as compared to negative control. Similar results were seenwith C14 thioesterase expression in the positive clones with an increaseRFE of about 60-fold to over 1200 fold increased expression as comparedto negative control.

A subset of the positive clones from each transformation (as screened bySouthern blotting and RT-qPCR assays) were selected and grown undernitrogen-replete conditions and analyzed for total lipid production andprofile. Lipid samples were prepared from dried biomass from each clone.20-40 mg of dried biomass from each transgenic clone was resuspended in2 mL of 3% H₂SO₄ in MeOH, and 200 ul of toluene containing anappropriate amount of a suitable internal standard (C19:0) was added.The mixture was sonicated briefly to disperse the biomass, then heatedat 65-70° C. for two hours. 2 mL of heptane was added to extract thefatty acid methyl esters, followed by addition of 2 mL of 6% K₂CO₃ (aq)to neutralize the acid. The mixture was agitated vigorously, and aportion of the upper layer was transferred to a vial containing Na₂SO₄(anhydrous) for gas chromatography analysis using standard FAME GC/FID(fatty acid methyl ester gas chromatography flame ionization detection)methods. Lipid profile (expressed as Area %) of the positive clones ascompared to wildtype negative control are summarized in Tables 36 and 37below. As shown in Table 36, the fold increase of C12 production in theC12 transformants ranged from about a 5-fold increase (clone C12-5) toover 11-fold increase (clone C12-1). Fold increase of C14 production inthe C14 transformants ranged from about a 1.5 fold increase to about a2.5 fold increase.

TABLE 36 Summary of total lipid profile of the Prototheca moriformis C12thioesterase transformants. Wildtype C12-1 C12-2 C12-3 C12-4 C12-5 C12-6C12-7 C12-8 C6:0 0.03 nd nd nd nd nd nd nd nd C8:0 0.11 0.09 nd 0.11 ndnd nd nd nd C10:0 nd nd nd 0.01 0.01 nd nd 0.01 nd C12:0 0.09 1.04 0.270.72 0.71 0.50 0.67 0.61 0.92 C14:0 2.77 2.68 2.84 2.68 2.65 2.79 2.732.56 2.69 C14:1 0.01 nd nd 0.02 nd nd nd 0.01 nd C15:0 0.30 0.09 0.100.54 0.19 0.09 0.13 0.97 0.09 C15:1 0.05 nd nd 0.02 nd nd nd nd nd C16:024.13  23.12  24.06  22.91  22.85  23.61  23.14  21.90  23.18  C16:10.57 0.62 0.10 0.52 0.69 0.63 0.69 0.49 0.63 C17:0 0.47 0.24 0.27 1.020.36 0.17 0.26 2.21 0.19 C17:1 0.08 nd 0.09 0.27 0.10 0.05 0.09 0.800.05 C18:0 nd nd 2.14 1.75 2.23 2.16 2.38 1.62 2.47 C18:1 22.10  23.15 24.61  21.90  23.52  19.30  22.95  20.22  22.85  C18:1 nd 0.33 0.24 ndnd 0.09 0.09 nd 0.11 C18:2 37.16  34.71  35.29  35.44  35.24  36.29 35.54  36.01  35.31  C18:3 11.68  11.29  9.26 11.62  10.76  13.61 10.64  11.97  10.81  alpha C20:0 0.15 0.16 0.19 0.16 0.16 0.14 0.18 0.140.18 C20:1 0.22 0.17 0.19 0.20 0.21 0.19 0.21 0.20 0.21 C20:2 0.05 nd0.04 0.05 0.05 0.05 0.04 0.05 0.04 C22:0 nd nd nd 0.01 nd nd nd 0.02 ndC22:1 nd nd nd nd nd 0.01 nd 0.01 nd C20:3 0.05 nd 0.07 0.06 0.06 0.100.07 0.05 0.06 C20:4 nd nd nd nd nd 0.02 nd nd nd C24:0 nd nd 0.24 0.010.20 0.19 0.19 0.14 0.20

TABLE 37 Summary of total lipid profile of the Prototheca moriformis C14thioesterase transformants. Wildtype C14-1 C14-2 C14-3 C14-4 C14-5 C14-6C14-7 C6:0 0.03 nd nd nd nd nd nd nd C8:0 0.11 nd nd nd nd nd nd ndC10:0 nd 0.01 nd 0.01 nd 0.01 nd nd C12:0 0.09 0.20 0.16 0.25 0.21 0.190.40 0.17 C14:0 2.77 4.31 4.76 4.94 4.66 4.30 6.75 4.02 C14:1 0.01 nd0.01 nd nd 0.01 nd nd C15:0 0.30 0.43 0.45 0.12 0.09 0.67 0.10 0.33C15:1 0.05 nd nd nd nd nd nd nd C16:0 24.13  22.85  23.20  23.83  23.84 23.48  24.04  23.34  C16:1 0.57 0.65 0.61 0.60 0.60 0.47 0.56 0.67 C17:00.47 0.77 0.76 0.21 0.19 1.11 0.18 0.54 C17:1 0.08 0.23 0.15 0.06 0.050.24 0.05 0.12 C18:0 nd 1.96 1.46 2.48 2.34 1.84 2.50 2.06 C18:1 22.10 22.25  19.92  22.36  20.57  19.50  20.63  22.03  C18:1 nd nd nd nd nd nd0.10 nd C18:2 37.16  34.97  36.11  34.35  35.70  35.49  34.03  35.60 C18:3 11.68  10.71  12.00  10.15  11.03  12.08  9.98 10.47  alpha C20:00.15 0.16 0.19 0.17 0.17 0.14 0.18 0.16 C20:1 0.22 0.20 0.12 .019 0.190.19 0.17 0.20 C20:2 0.05 0.04 0.02 0.03 0.04 0.05 0.03 0.04 C22:0 nd ndnd nd 0.02 0.01 nd nd C22:1 nd 0.01 nd nd nd nd nd 0.01 C20:3 0.05 0.080.03 0.06 0.09 0.05 0.05 0.07 C20:4 nd 0.01 nd nd nd nd 0.02 nd C24:0 nd0.17 0.14 0.19 0.20 0.16 0.22 0.17

The above-described experiments indicate the successful transformationof Prototheca moriformis (UTEX 1435) with transgene constructs of twodifferent thioesterases (C12 and C14), which involved not only thesuccessful expression of the transgene, but also the correct targetingof the expressed protein to the plastid and a functional effect (theexpected change in lipid profile) as a result of the transformation. Thesame transformation experiment was performed using an expressionconstruct containing a codon-optimized (according to Table 4) Cupheahookeriana C8-10 thioesterase coding region with the native plastidtargeting sequence (SEQ ID NO: 70) yielded no change in lipid profile.While the introduction of the Cuphea hookeriana C8-10 transgene intoPrototheca moriformis (UTEX 1435) was successful and confirmed bySouthern blot analysis, no change in C8 or C10 fatty acid production wasdetected in the transformants compared to the wildtype strain.

Example 11 Generation of Prototheca moriformis Strain with ExogenousPlant TE with Algal Plastid Targeting Sequence

In order to investigate whether the use of algal chloroplast/plastidtargeting sequences would improve medium chain (C8-C14) thioesteraseexpression and subsequent medium chain lipid production in Protothecamoriformis (UTEX 1435), several putative algal plastid targetingsequences were cloned from Chlorella protothecoides and Protothecamoriformis. Thioesterase constructs based on Cuphea hookeriana C8-10thioesterase, Umbellularia californica C12 thioesterase, and Cinnamomumcamphora C14 thioesterase were made using made with a Chlorellasorokiniana glutamate dehydrogenase promoter/5′UTR and a Chlorellavulgaris nitrate reductase 3′UTR. The thioesterase coding sequences weremodified by removing the native plastid targeting sequences andreplacing them with plastid targeting sequences from the Chlorellaprotothecoides and the Prototheca moriformis genomes. The thioesteraseexpression constructs and their corresponding sequence identificationnumbers are listed below. Each transformation plasmid also contained aNeo resistance construct that was identical to the ones described inExample 9 above. Additionally, another algal-derived promoter, theChlamydomonas reinhardtii β-tubulin promoter, was also tested inconjunction with the thioesterase constructs. “Native” plastid targetingsequence refers to the higher plant thioesterase plastid targetingsequence. A summary of the constructs used in these experiments isprovided below:

Construct Promoter/ Plastid Name 5′UTR targeting seq Gene 3′UTR SEQ IDNO. Construct 1 C. sorokiniana C. protothecoides Cuphea C. vulgaris SEQID NO: 71 glutamate stearoyl ACP hookeriana nitrate dehydrogenasedesaturase C8-10 TE reductase Construct 2 C. sorokiniana P. moriformisCuphea C. vulgaris SEQ ID NO: 72 glutamate delta 12 fatty hookeriananitrate dehydrogenase acid desaturase C8-10 TE reductase Construct 3 C.sorokiniana P. moriformis Cuphea C. vulgaris SEQ ID NO: 73 glutamateisopentenyl hookeriana nitrate dehydrogenase diphosphate C8-10 TEreductase synthase Construct 4 C. sorokiniana P. moriformis UmbellulariaC. vulgaris SEQ ID NO: 74 glutamate isopentenyl californica nitratedehydrogenase diphosphate C12 TE reductase synthase Construct 5 C.sorokiniana P. moriformis Umbellularia C. vulgaris SEQ ID NO: 75glutamate stearoyl ACP californica nitrate dehydrogenase desaturase C12TE reductase Construct 6 C. sorokiniana C. protothecoides UmbellulariaC. vulgaris SEQ ID NO: 76 glutamate stearoyl ACP californica nitratedehydrogenase desaturase C12 TE reductase Construct 7 C. sorokiniana P.moriformis Umbellularia C. vulgaris SEQ ID NO: 77 glutamate delta 12fatty californica nitrate dehydrogenase acid desaturase C12 TE reductaseConstruct 8 C. sorokiniana C. protothecoides Cinnamomum C. vulgaris SEQID NO: 78 glutamate stearoyl ACP camphora nitrate dehydrogenasedesaturase C14 TE reductase Construct 9 Chlamydomonas Native Cuphea C.vulgaris SEQ ID NO: 79 reinhardtii hookeriana nitrate β-tubulin C8-10 TEreductase Construct 10 Chlamydomonas P. moriformis Cuphea C. vulgarisSEQ ID NO: 80 reinhardtii isopentenyl hookeriana nitrate β-tubulindiphosphate C8-10 TE reductase synthase Construct 11 Chlamydomonas P.moriformis Cuphea C. vulgaris SEQ ID NO: 81 reinhardtii delta 12 fattyhookeriana nitrate β-tubulin acid desaturase C8-10 TE reductaseConstruct 12 Chlamydomonas C. protothecoides Cuphea C. vulgaris SEQ IDNO: 82 reinhardtii stearoyl ACP hookeriana nitrate β-tubulin desaturaseC8-10 TE reductase Construct 13 Chlamydomonas P. moriformis Cuphea C.vulgaris SEQ ID NO: 83 reinhardtii stearoyl ACP hookeriana nitrateβ-tubulin desaturase C8-10 TE reductase Construct 14 ChlamydomonasNative Umbellularia C. vulgaris SEQ ID NO: 84 reinhardtii californicanitrate β-tubulin C12 TE reductase Construct 15 Chlamydomonas P.moriformis Umbellularia C. vulgaris SEQ ID NO: 85 reinhardtiiisopentenyl californica nitrate β-tubulin diphosphate C12 TE reductaseConstruct 16 Chlamydomonas P. moriformis Umbellularia C. vulgaris SEQ IDNO: 86 reinhardtii delta 12 fatty californica nitrate β-tubulin aciddesaturase C12 TE reductase Construct 17 Chlamydomonas C. protothecoidesUmbellularia C. vulgaris SEQ ID NO: 87 reinhardtii stearoyl ACPcalifornica nitrate β-tubulin desaturase C12 TE reductase Construct 18Chlamydomonas P. moriformis Umbellularia C. vulgaris SEQ ID NO: 88reinhardtii stearoyl ACP californica nitrate β-tubulin desaturase C12 TEreductase Construct 19 Chlamydomonas Native Cinnamomum C. vulgaris SEQID NO: 89 reinhardtii camphora nitrate β-tubulin C14 TE reductaseConstruct 20 Chlamydomonas P. moriformis Cinnamomum C. vulgaris SEQ IDNO: 90 reinhardtii isopentenyl camphora nitrate β-tubulin diphosphateC14 TE reductase synthase Construct 21 Chlamydomonas P. moriformisCinnamomum C. vulgaris SEQ ID NO: 91 reinhardtii delta 12 fatty camphoranitrate β-tubulin acid desaturase C14 TE reductase Construct 22Chlamydomonas C. protothecoides Cinnamomum C. vulgaris SEQ ID NO: 92reinhardtii stearoyl ACP camphora nitrate β-tubulin desaturase C14 TEreductase Construct 23 Chlamydomonas P. moriformis Cinnamomum C.vulgaris SEQ ID NO: 93 reinhardtii stearoyl ACP camphora nitrateβ-tubulin desaturase C14 TE reductase

Each construct was transformed into Prototheca moriformis (UTEX 1435)and selection was performed using G418 using the methods described inExample 9 above. Several positive clones from each transformation werepicked and screened for the presence thioesterase transgene usingSouthern blotting analysis. Expression of the thioesterase transgene wasconfirmed using RT-PCR. A subset of the positive clones (as confirmed bySouthern blotting analysis and RT-PCR) from each transformation wasselected and grown for lipid profile analysis. Lipid samples wereprepared from dried biomass samples of each clone and lipid profileanalysis was performed using acid hydrolysis methods described inExample 9. Changes in area percent of the fatty acid corresponding tothe thioesterase transgene were compared to wildtype levels, and clonestransformed with a thioesterase with the native plastid targetingsequence.

The clones transformed with Cuphea hookeriana C8-10 thioesteraseconstructs with the native plastid targeting sequence had the same levelof C8 and C10 fatty acids as wildtype. The clones transformed withCuphea hookeriana C8-10 thioesterase constructs (Constructs 1-3) withalgal plastid targeting sequences had over a 10-fold increase in C10fatty acids for Construct 3 and over 40-fold increase in C10 fatty acidsfor Constructs 1 and 2 (as compared to wildtype). The clones transformedwith Umbellularia californica C12 thioesterase constructs with thenative plastid targeting sequence had a modest 6-8 fold increase in C12fatty acid levels as compared to wildtype. The clones transformed withthe Umbellularia californica C12 thioesterase constructs with the algalplasid targeting constructs (Constructs 4-7) had over an 80-foldincrease in C12 fatty acid level for Construct 4, about an 20-foldincrease in C12 fatty acid level for Construct 6, about a 10-foldincrease in C12 fatty acid level for Construct 7 and about a 3-foldincrease in C12 fatty acid level for Construct 5 (all compared towildtype). The clones transformed with Cinnamomum camphora C14thioesterase with either the native plastid targeting sequence or theconstruct 8 (with the Chlorella protothecoides stearoyl ACP desaturaseplastid targeting sequence) had about a 2-3 fold increase in C14 fattyacid levels as compared to wildtype. In general clones transformed withan algal plastid targeting sequence thioesterase constructs had a higherfold increase in the corresponding chain-length fatty acid levels thanwhen using the native higher plant targeting sequence.

1. Clamydomonas reinhartii O-Tubulin Promoter

Additional heterologous thioesterase expression constructs were preparedusing the Chlamydomonas reinhardtii β-tubulin promoter instead of the C.sorokinana glutamate dehydrogenase promoter. The construct elements andsequence of the expression constructs are listed above. Each constructwas transformed into Prototheca moriformis UTEX 1435 host cells usingthe methods described above. Lipid profiles were generated from a subsetof positive clones for each construct in order to assess the success andproductivity of each construct. The lipid profiles compare the fattyacid levels (expressed in area %) to wildtype host cells. The “Mean”column represents the numerical average of the subset of positiveclones. The “Sample” column represents the best positive clone that wasscreened (best being defined as the sample that produced the greatestchange in area % of the corresponding chain-length fatty acidproduction). The “low-high” column represents the lowest area % and thehighest area % of the fatty acid from the clones that were screened. Thelipid profile results of Constructs 9-12 and 14-23 are summarized below.

Construct 9. Cuphea hookeriana C8-10 TE

Fatty Acid wildtype Mean Sample low/high C 8:0 0 0.05 0.30 0-0.29 C 10:00.01 0.63 2.19 0-2.19 C 12:0 0.03 0.06 0.10 0-0.10 C 14:0 1.40 1.50 1.411.36-3.59   C 16:0 24.01 24.96 24.20 C 16:1 0.67 0.80 0.85 C 17:0 0 0.160.16 C 17:1 0 0.91 0 C 18:0 4.15 17.52 3.19 C 18:1 55.83 44.81 57.54 C18:2 10.14 7.58 8.83 C 18:3α 0.93 0.68 0.76 C 20:0 0.33 0.21 0.29 C 24:00 0.05 0.11Construct 10. Cuphea hookeriana C8-10 TE

Fatty Acid wildtype Mean Sample low/high C 8:0 0 0.01 0.02 0-0.03 C 10:00 0.16 0.35 0-0.35 C 12:0 0.04 0.05 0.07 0-0.07 C 14:0 1.13 1.62 1.810-0.05 C 14:1 0 0.04 0.04 C 15:0 0.06 0.05 0.05 C 16:0 19.94 26.42 28.08C 16:1 0.84 0.96 0.96 C 17:0 0.19 0.14 0.13 C 17:1 0.10 0.06 0.05 C 18:02.68 3.62 3.43 C 18:1 63.96 54.90 53.91 C 18:2 9.62 9.83 9.11 C 18:3 γ 00.01 0 C 18:3α 0.63 0.79 0.73 C 20:0 0.26 0.35 0.33 C 20:1 0.06 0.080.09 C 20:1 0.08 0.06 0.07 C 22:0 0 0.08 0.09 C 24:0 0.13 0.13 0.11Construct 11. Cuphea hookeriana C8-10 TE

Fatty Acid wildtype Mean Sample low/high C 8:0 0 0.82 1.57   0-1.87 C10:0 0 3.86 6.76   0-6.76 C 12:0 0.04 0.13 0.20 0.03-0.20 C 14:0 1.131.80 1.98 1.64-2.05 C 14:1 0 0.04 0.04 C 15:0 0.06 0.06 0.06 C 16:019.94 25.60 25.44 C 16:1 0.84 1.01 1.02 C 17:0 0.19 0.13 0.11 C 17:10.10 0.06 0.05 C 18:0 2.68 2.98 2.38 C 18:1 63.96 51.59 48.85 C 18:29.62 9.85 9.62 C 18:3 γ 0 0.01 0 C 18:3α 0.63 0.91 0.92 C 20:0 0.26 0.290.26 C 20:1 0.06 0.06 0 C 20:1 0.08 0.06 0.03 C 22:0 0 0.08 0.08 C 24:00.13 0.06 0Construct 12. Cuphea hookeriana C8-10 TE

Fatty Acid wildtype Mean Sample low/high C 8:0 0 0.31 0.85   0-0.85 C10:0 0 2.16 4.35 0.20-4.35 C 12:0 0.04 0.10 0.15   0-0.18 C 14:0 1.131.96 1.82 1.66-2.97 C 14:1 0 0.03 0.04 C 15:0 0.06 0.07 0.07 C 16:019.94 26.08 25.00 C 16:1 0.84 1.04 0.88 C 17:0 0.19 0.16 0.16 C 17:10.10 0.05 0.07 C 18:0 2.68 3.02 3.19 C 18:1 63.96 51.08 52.15 C 18:29.62 11.44 9.47 C 18:3 γ 0 0.01 0 C 18:3α 0.63 0.98 0.90 C 20:0 0.260.30 0.28 C 20:1 0.06 0.06 0.05 C 20:1 0.08 0.04 0 C 22:0 0 0.07 0 C24:0 0.13 0.05 0Construct 14. Umbellularia californica C12 TE

Fatty Acid wildtype Mean Sample low/high C 10:0 0.01 0.02 0.03 0.02-0.03C 12:0 0.03 2.62 3.91 0.04-3.91 C 14:0 1.40 1.99 2.11 1.83-2.19 C 16:024.01 27.64 27.01 C 16:1 0.67 0.92 0.92 C 18:0 4.15 2.99 2.87 C 18:155.83 53.22 52.89 C 18:2 10.14 8.68 8.41 C 18:3α 0.93 0.78 0.74 C 20:00.33 0.29 0.27Construct 15. Umbellularia californica C12 TE

Fatty Acid wildtype Mean Sample low/high C 10:0 0 0.05 0.08   0-0.08 C12:0 0.04 8.12 12.80  4.35-12.80 C 13:0 0 0.02 0.03   0-0.03 C 14:0 1.132.67 3.02 2.18-3.37 C 14:1 0 0.04 0.03 0.03-0.10 C 15:0 0.06 0.07 0.06 C16:0 19.94 25.26 23.15 C 16:1 0.84 0.99 0.86 C 17:0 0.19 0.14 0.14 C17:1 0.10 0.05 0.05 C 18:0 2.68 2.59 2.84 C 18:1 63.96 46.91 44.93 C18:2 9.62 10.59 10.01 C 18:3α 0.63 0.92 0.83 C 20:0 0.26 0.27 0.24 C20:1 0.06 0.06 0.06 C 20:1 0.08 0.05 0.04 C 22:0 0 0.07 0.09 C 24:0 0.130.13 0.12Construct 16. Umbellularia californica C12 TE

Fatty Acid wildtype Mean Sample low/high C 10:0 0 0.03 0.04 0.02-0.04 C12:0 0.04 2.43 5.32 0.98-5.32 C 13:0 0 0.01 0.02   0-0.02 C 14:0 1.131.77 1.93 1.62-1.93 C 14:1 0 0.03 0.02 0.02-0.04 C 15:0 0.06 0.06 0.05 C16:0 19.94 24.89 22.29 C 16:1 0.84 0.91 0.82 C 17:0 0.19 0.16 0.15 C17:1 0.10 0.06 0.06 C 18:0 2.68 3.81 3.67 C 18:1 63.96 53.19 52.82 C18:2 9.62 10.38 10.57 C 18:3α 0.63 0.80 0.77 C 20:0 0.26 0.35 0.32 C20:1 0.06 0.06 0.07 C 20:1 0.08 0.07 0.08 C 22:0 0 0.08 0.07 C 24:0 0.130.15 0.14Construct 17. Umbellularia californica C12 TE

Fatty Acid wildtype Mean Sample low/high C 10:0 0 0.04 0.07 0.03-0.08 C12:0 0.04 7.02 14.11  4.32-14.11 C 13:0 0 0.03 0.04 0.01-0.04 C 14:01.13 2.25 3.01 1.95-3.01 C 14:1 0 0.03 0.03 0.02-0.03 C 15:0 0.06 0.060.06 C 16:0 19.94 23.20 21.46 C 16:1 0.84 0.82 0.77 C 17:0 0.19 0.150.14 C 17:1 0.10 0.06 0.06 C 18:0 2.68 3.47 2.93 C 18:1 63.96 50.3045.17 C 18:2 9.62 10.33 9.98 C 18:3 γ 0 0.01 0 C 18:3α 0.63 0.84 0.86 C20:0 0.26 0.32 0.27 C 20:1 0.06 0.07 0.06 C 20:1 0.08 0.06 0.06 C 22:0 00.08 0.09 C 24:0 0.13 0.14 0.13Construct 18. Umbellularia californica C12 TE

Fatty Acid wildtype Mean Sample low/high C 10:0 0 0.03 0.05 0.01-0.05 C12:0 0.04 5.06 7.77 0.37-7.77 C 13:0 0 0.02 0   0-0.03 C 14:0 1.13 2.112.39 1.82-2.39 C 14:1 0 0.03 0.03 0.02-0.05 C 15:0 0.06 0.06 0.06 C 16:019.94 24.60 23.95 C 16:1 0.84 0.86 0.83 C 17:0 0.19 0.15 0.14 C 17:10.10 0.06 0.05 C 18:0 2.68 3.31 2.96 C 18:1 63.96 51.26 49.70 C 18:29.62 10.18 10.02 C 18:3 γ 0 0.01 0.02 C 18:3α 0.63 0.86 0.86 C 20:0 0.260.32 0.29 C 20:1 0.06 0.05 0.05 C 20:1 0.08 0.07 0.04 C 22:0 0 0.08 0.08C 24:0 0.13 0.13 0.13Construct 19. Cinnamomum camphora C14 TE

Fatty Acid wildtype Mean Sample low/high C 10:0 0.02 0.01 0.01 0.01-0.02C 12:0 0.05 0.27 0.40 0.08-0.41 C 14:0 1.52 4.47 5.81 2.10-5.81 C 16:025.16 28.14 28.55 C 16:1 0.72 0.84 0.82 C 18:0 3.70 3.17 2.87 C 18:154.28 51.89 51.01 C 18:2 12.24 9.36 8.62 C 18:3α 0.87 0.74 0.75 C 20:00.33 0.33 0.31Construct 20. Cinnamomum camphora C14 TE

Fatty Acid wildtype Mean Sample low/high C 10:0 0.01 0.01 0.02 0.01-0.02C 12:0 0.03 0.39 0.65 0.08-0.65 C 13:0 0 0.01 0.01 0.01-0.02 C 14:0 1.405.61 8.4 2.1-8.4 C 14:1 0 0.03 0.03 0.02-0.03 C 15:0 0 0.06 0.07 C 16:024.01 25.93 25.57 C 16:1 0.67 0.75 0.71 C 17:0 0 0.13 0.12 C 17:1 0 0.050.05 C 18:0 4.15 3.30 3.23 C 18:1 55.83 51.00 48.48 C 18:2 10.14 10.3810.35 C 18:3α 0.93 0.91 0.88 C 20:0 0.33 0.35 0.32 C 20:1 0 0.08 0.08 C20:1 0 0.07 0.07 C 22:0 0 0.08 0.08 C 24:0 0 0.14 0.13Construct 21. Cinnamomum camphora C14 TE

Fatty Acid wildtype Mean Sample low/high C 10:0 0.01 0.01 0.01   0-0.01C 12:0 0.03 0.10 0.27 0.04-0.27 C 14:0 1.40 2.28 4.40 1.47-4.40 C 16:024.01 26.10 26.38 C 16:1 0.67 0.79 0.73 C 17:0 0 0.15 0.16 C 17:1 0 0.060.06 C 18:0 4.15 3.59 3.51 C 18:1 55.83 53.53 50.86 C 18:2 10.14 10.8311.11 C 18:3α 0.93 0.97 0.87 C 20:0 0.33 0.36 0.37 C 20:1 0 0.09 0.08 C20:1 0 0.07 0.07 C 22:0 0 0.09 0.09Construct 22. Cinnamomum camphora C14 TE

Fatty Acid wildtype Mean Sample low/high C 10:0 0.01 0.02 0.02 0.02-0.02C 12:0 0.03 1.22 1.83 0.59-1.83 C 13:0 0 0.02 0.03 0.01-0.03 C 14:0 1.4012.77 17.33  7.97-17.33 C 14:1 0 0.02 0.02 0.02-0.04 C 15:0 0 0.07 0.08C 16:0 24.01 24.79 24.22 C 16:1 0.67 0.64 0.58 C 17:0 0 0.11 0.10 C 17:10 0.04 0.04 C 18:0 4.15 2.85 2.75 C 18:1 55.83 45.16 41.23 C 18:2 10.149.96 9.65 C 18:3α 0.93 0.91 0.85 C 20:0 0.33 0.30 0.30 C 20:1 0 0.070.06 C 20:1 0 0.06 0.05 C 22:0 0 0.08 0.08Construct 23. Cinnamomum camphora C14 TE

Fatty Acid wildtype Mean Sample low/high C 10:0 0.01 0.01 0.02   0-0.02C 12:0 0.05 0.57 1.08 0.16-1.08 C 13:0 0 0.02 0.02   0-0.02 C 14:0 1.457.18 11.24  2.96-11.24 C 14:1 0.02 0.03 0.03 0.02-0.03 C 15:0 0.06 0.070.07 C 16:0 24.13 25.78 25.21 C 16:1 0.77 0.72 0.66 C 17:0 0.19 0.130.11 C 17:1 0.08 0.05 0.04 C 18:0 3.53 3.35 3.12 C 18:1 56.15 49.6546.35 C 18:2 11.26 10.17 9.72 C 18:3α 0.84 0.95 0.83 C 20:0 0.32 0.340.32 C 20:1 0.09 0.08 0.09 C 20:1 0.07 0.05 0.06 C 22:0 0.07 0.08 0.08 C24:0 0.13 0.13 0.12

Constructs 9-12 were expression vectors containing the Cuphea hookerianaC8-10 thioesterase construct. As can be seen in the data summariesabove, the best results were seen with Construct 11, with the Sample C8fatty acid being 1.57 Area % (as compared to 0 in wildtype) and C10fatty acid being 6.76 Area % (as compared to 0 in wildtype). There wasalso a modest increase in C12 fatty acids (approximately 2-5 foldincrease). While the native plastid targeting sequence produced nochange when under the control of the C. sorokinana glutamatedehydrogenase promoter, the same expression construct driven by the C.reinhardtii β-tubulin promoter produced significant changes in C8-10fatty acids in the host cell. This is further evidence of theidiosyncrasies of heterologous expression of thioesterases in Protothecaspecies. All of the clones containing the C. reinhardtii β-tubulinpromoter C8-10 thioesterase construct had greater increases in C8-10fatty acids than the clones containing the C. sorokinana glutamatedehydrogenase promoter C8-10 thioesterase construct. Lipid profile datafor Construct 13 was not obtained and therefore, not included above.

Constructs 14-18 were expression vectors containing the Umbellulariacalifornica C12 thioesterase construct. As can be seen in the datasummaries above, the best results were seen with Constructs 15 (P.moriformis isopentenyl diphosphate synthase plastid targeting sequence)and 17 (C. protothecoides stearoyl ACP desaturase plastid targetingsequence). The greatest change in C12 fatty acid production was seenwith Construct 17, with C12 fatty acids levels of up to 14.11 area %, ascompared to 0.04 area % in wildtype. Modest changes (about 2-fold) werealso seen with C14 fatty acid levels. When compared to the sameconstructs with the C. sorokinana glutamate dehydrogenase promoter, thesame trends were true with the C. reinhardtii β-tubulin promoter—the C.protothecoides stearoyl ACP desaturase and P.monjormis isopenienyiutpnospnate syntnase piasuu targeting sequences prouuceo me greatestchange in C12 faily acid levels with both promoters.

Constructs 19-23 were expression vectors containing the Cinnamomumcamp/torn CM ihuvsicrase construct. As can W seen in ihe data summariesabove, tbe besl results were seen with Constructs 22 and Construct 23.The greatest change in C14 Tatty acid production was seen with Construct22. with C14 fatty acid levels of up to 17.35 area % (when the valuesforC140and C141 are combined), as compared to 1.40% in wildlype. Changesin C12 fatly acids were also seen (5-60 fold). When compared to the sameconstrucis with the C.wrokinaiui glutamate dehydrogenase promoter, thesame trends were true with ihe C.rcinhurdtii p-tubulin promoter-theC.protoihecoides slearoyl AO* desaturase and P.moriformis slearoyl ACTdesaturase plaslid targeting sequences produced the greatest change inC14 fatly acid levels with both promoters. Consistently wilh allihioeslerase expression constructs, ihe Creinhardtii p-iubulin promoterconstructs produced greater changes in C8-14 fatly acid levels than iheC.sorokiniana gluiamate dehydrogenase

Two positive clones from the Construct 22 were selected and grown underhigh selective pressure (50mg/l, C1418). After 6 days in culture, theclones were harvested and their lipid profile was determined using themethods described above. Ilie lipid profile data is summarized below andis expressed in area %.

Construct 22 clones + 50 mg/L G418 Fatty Acid Construct 22 A Construct22 B C 12:0 3.21 3.37 C 14:0 27.55 26.99 C 16:0 25.68 24.37 C 16:1 0.990.92 C 18:0 1.37 1.23 C 18:1 28.35 31.07 C 18:2 11.73 11.05 C 18:3α 0.920.81 C 20:0 0.16 0.17

Both clones, when grown under constant, high selective pressure,produced an increased amount of CI4 and C12 fatty acids, about doublethe levels seen with Construct 22 above. These clones yielded over 30area of C12-14 fatty acids, as compared to 1.5 area % of C12-14 fattyacids seen in wildlype cells.

1-36. (canceled)
 37. A method of extracting a lipid from recombinantPrototheca cells, the method comprising the steps of: (a) lysingrecombinant Prototheca cells to produce a lysate, wherein therecombinant Prototheca cells: (i) have not been subjected to a dryingstep between culturing and lysing; and (ii) comprise at least 10% lipidby dry weight, and wherein the fatty acid profile of the lipid is atleast 10% C8-C14; (b) treating the lysate with an organic solvent for aperiod of time; (c) separating the treated lysate into layers comprisinga lipid layer and an aqueous layer and, optionally, a lipid:aqueousemulsion layer, and/or a cell pellet; and (d) removing the lipid fromthe other layer(s).
 38. The method of claim 37, wherein step (d) furthercomprises reducing the temperature of the mixture to below 25° C. 39.The method of claim 37, wherein the recombinant Prototheca cellscomprise lipid that is at least 10% C14 triacylglycerols, at least 10%C12 triacylglycerols, or at least 10% C10 triacylglycerols.
 40. A methodof producing fuel comprising the steps of: (a) lysing recombinantPrototheca cells to produce a lysate, wherein the recombinant Protothecacells: (i) have not been subjected to a drying step between culturingand lysing; and (ii) comprises at least 10% lipid by dry weight, andwherein the fatty acid profile of the lipid is at least 10% C8-C14; (b)treating the lysate with an organic solvent for a period of time; (c)separating the treated lysate into layers comprising a lipid layer andan aqueous layer and, optionally, a lipid:aqueous emulsion layer, and/orcell pellet; (d) removing the lipid from the other layer(s); and (e)subjecting the lipid to a chemical reaction, to produce the fuel. 41.The method of claim 40, wherein the fuel is biodiesel, renewable diesel,or jet fuel.
 42. The method of claim 40, wherein the chemical reactionis one or more chemical reactions selected from transesterification,hydroprocessing, deoxygenation, isomerization, hydrotreating,hydrolysis, or fluid catalytic cracking.
 43. The method of claim 41,wherein the fuel is biodiesel that meets or exceeds the ASTM D6751biodiesel standard or a biodiesel that meets or exceeds the EN 14214biodiesel standard.
 44. The method of claim 41, wherein the fuel isrenewable diesel that meets or exceeds the ASTM D 975 standard.
 45. Themethod of claim 41, wherein the fuel is jet fuel.
 46. The method ofclaim 37, wherein the organic solvent is methanol.
 47. The method ofclaim 37, wherein the lysing is accomplished by subjecting therecombinant Prototheca cells to heating, sonication, mechanical lysis,osmotic shock, expression of an autolysis gene, exposure to pH above 8,exposure to an acidic pH, heating and exposure to an acidic pH, ordigestion with an enzyme.
 48. The method of claim 47, wherein the lysingis accomplished by subjecting the recombinant Prototheca cells tosonication.
 49. The method of claim 37, wherein the recombinantPrototheca cells comprise a polynucleotide encoding a sucrose invertaseor a lipid pathway enzyme.
 50. The method of claim 49, wherein thepolynucleotide encodes a lipid pathway enzyme.
 51. The method of claim49, wherein the recombinant Prototheca cells comprise one or morepolynucleotides encoding a sucrose invertase and a lipid pathway enzyme.52. The method of claim 50, wherein the lipid pathway enzyme is a fattyacyl-ACP thioesterase.
 53. The method of claim 40, wherein the organicsolvent is methanol.
 54. The method of claim 40, wherein the lysing isaccomplished by subjecting the recombinant Prototheca cells to heating,sonication, mechanical lysis, osmotic shock, expression of an autolysisgene, exposure to pH above 8, exposure to an acidic pH, heating andexposure to an acidic pH, or digestion with an enzyme.
 55. The method ofclaim 40, wherein the recombinant Prototheca cells comprise apolynucleotide encoding a sucrose invertase or a lipid pathway enzyme.56. The method of claim 51, wherein the lipid pathway enzyme is a fattyacyl-ACP thioesterase.